Wednesday, November 30, 2011
Egypt Ellection
Iam really very happy for the ellection in Egypt. Iam near fivty years old and I did not see free ellection in my country before. Sorry Ican not now write in microbiology as my mind is really occupied. Iwill writ again, but now it is the time of my country.
Tuesday, November 29, 2011
Monday, November 28, 2011
Pneumonia
Pneumonia is an important cause of morbidity and mortality in adults and children. It results from the host inflammatory response to infection of the distal airways and the lung alveoli (Schutter et.al, 2011). The most useful classification is based on the site of acquisition: community-acquired (CAP) or hospital-acquired pneumonia (HAP) (Esperatti and Marti, 2008).
Hospital-associated pneumonia (HAP) is the second most common nosocomial infection (after urinary tract infection) and the most common nosocomial infection acquired in the intensive care unit (ICU). HAP associated with mechanical ventilation is called ventilator-associated pneumonia (VAP) (Franzetti et.al., 2010). VAP has an estimated incidence of 8–28% and is associated with an excess of ICU stay, increased costs, and attributable mortality (Safdar et.al., 2005).
The term atypical pneumonia was originally used to describe an unusual presentation of pneumonia. It is now more widely used in reference to either pneumonia caused by a relatively common group of pathogens, or to a distinct clinical syndrome the existence of which is difficult to demonstrate (Murdoch and Chambers, 2009).
The “atypical pathogen” group includes Mycoplasma pneumoniae; Legionella; Chlamydia pneumoniae; Coxiella burnettii; and the respiratory viruses, especially
Introduction and Aim of the Work
influenza A and B, parainfluenza 1, 2, and 3, respiratory syncytial virus (RSV) and Epstein-Barr virus (EBV) (Lieberman, 2005).
The diagnosis of pneumonia in the hospitalized patient is even more challenging than the diagnosis of CAP (Richards et.al., 2000). Clinical findings alone are not sufficient for a definitive diagnosis. Therfore, a variety of noninvasive and invasive tests have been proposed as guides for diagnosis and treatment of hospital-acquired pneumonia. Methods include sputum Gram stain and culture, serologic studies, antigen detection tests, and nucleic acid amplification methods (Carroll, 2002).
Many patient- and disease-specific factors contribute to the pathophysiology of HAP, particularly in the surgical population. Risk-factor modification and inpatient prevention strategies can have a significant impact on the incidence of HAP (Kieninger and Lipsett, 2009).
Hospital-associated pneumonia (HAP) is the second most common nosocomial infection (after urinary tract infection) and the most common nosocomial infection acquired in the intensive care unit (ICU). HAP associated with mechanical ventilation is called ventilator-associated pneumonia (VAP) (Franzetti et.al., 2010). VAP has an estimated incidence of 8–28% and is associated with an excess of ICU stay, increased costs, and attributable mortality (Safdar et.al., 2005).
The term atypical pneumonia was originally used to describe an unusual presentation of pneumonia. It is now more widely used in reference to either pneumonia caused by a relatively common group of pathogens, or to a distinct clinical syndrome the existence of which is difficult to demonstrate (Murdoch and Chambers, 2009).
The “atypical pathogen” group includes Mycoplasma pneumoniae; Legionella; Chlamydia pneumoniae; Coxiella burnettii; and the respiratory viruses, especially
Introduction and Aim of the Work
influenza A and B, parainfluenza 1, 2, and 3, respiratory syncytial virus (RSV) and Epstein-Barr virus (EBV) (Lieberman, 2005).
The diagnosis of pneumonia in the hospitalized patient is even more challenging than the diagnosis of CAP (Richards et.al., 2000). Clinical findings alone are not sufficient for a definitive diagnosis. Therfore, a variety of noninvasive and invasive tests have been proposed as guides for diagnosis and treatment of hospital-acquired pneumonia. Methods include sputum Gram stain and culture, serologic studies, antigen detection tests, and nucleic acid amplification methods (Carroll, 2002).
Many patient- and disease-specific factors contribute to the pathophysiology of HAP, particularly in the surgical population. Risk-factor modification and inpatient prevention strategies can have a significant impact on the incidence of HAP (Kieninger and Lipsett, 2009).
Pneumonia
Pneumonia is an important cause of morbidity and mortality in adults and children. It results from the host inflammatory response to infection of the distal airways and the lung alveoli (Schutter et.al, 2011). The most useful classification is based on the site of acquisition: community-acquired (CAP) or hospital-acquired pneumonia (HAP) (Esperatti and Marti, 2008).
Hospital-associated pneumonia (HAP) is the second most common nosocomial infection (after urinary tract infection) and the most common nosocomial infection acquired in the intensive care unit (ICU). HAP associated with mechanical ventilation is called ventilator-associated pneumonia (VAP) (Franzetti et.al., 2010). VAP has an estimated incidence of 8–28% and is associated with an excess of ICU stay, increased costs, and attributable mortality (Safdar et.al., 2005).
The term atypical pneumonia was originally used to describe an unusual presentation of pneumonia. It is now more widely used in reference to either pneumonia caused by a relatively common group of pathogens, or to a distinct clinical syndrome the existence of which is difficult to demonstrate (Murdoch and Chambers, 2009).
The “atypical pathogen” group includes Mycoplasma pneumoniae; Legionella; Chlamydia pneumoniae; Coxiella burnettii; and the respiratory viruses, especially
Introduction and Aim of the Work
influenza A and B, parainfluenza 1, 2, and 3, respiratory syncytial virus (RSV) and Epstein-Barr virus (EBV) (Lieberman, 2005).
The diagnosis of pneumonia in the hospitalized patient is even more challenging than the diagnosis of CAP (Richards et.al., 2000). Clinical findings alone are not sufficient for a definitive diagnosis. Therfore, a variety of noninvasive and invasive tests have been proposed as guides for diagnosis and treatment of hospital-acquired pneumonia. Methods include sputum Gram stain and culture, serologic studies, antigen detection tests, and nucleic acid amplification methods (Carroll, 2002).
Many patient- and disease-specific factors contribute to the pathophysiology of HAP, particularly in the surgical population. Risk-factor modification and inpatient prevention strategies can have a significant impact on the incidence of HAP (Kieninger and Lipsett, 2009).
Hospital-associated pneumonia (HAP) is the second most common nosocomial infection (after urinary tract infection) and the most common nosocomial infection acquired in the intensive care unit (ICU). HAP associated with mechanical ventilation is called ventilator-associated pneumonia (VAP) (Franzetti et.al., 2010). VAP has an estimated incidence of 8–28% and is associated with an excess of ICU stay, increased costs, and attributable mortality (Safdar et.al., 2005).
The term atypical pneumonia was originally used to describe an unusual presentation of pneumonia. It is now more widely used in reference to either pneumonia caused by a relatively common group of pathogens, or to a distinct clinical syndrome the existence of which is difficult to demonstrate (Murdoch and Chambers, 2009).
The “atypical pathogen” group includes Mycoplasma pneumoniae; Legionella; Chlamydia pneumoniae; Coxiella burnettii; and the respiratory viruses, especially
Introduction and Aim of the Work
influenza A and B, parainfluenza 1, 2, and 3, respiratory syncytial virus (RSV) and Epstein-Barr virus (EBV) (Lieberman, 2005).
The diagnosis of pneumonia in the hospitalized patient is even more challenging than the diagnosis of CAP (Richards et.al., 2000). Clinical findings alone are not sufficient for a definitive diagnosis. Therfore, a variety of noninvasive and invasive tests have been proposed as guides for diagnosis and treatment of hospital-acquired pneumonia. Methods include sputum Gram stain and culture, serologic studies, antigen detection tests, and nucleic acid amplification methods (Carroll, 2002).
Many patient- and disease-specific factors contribute to the pathophysiology of HAP, particularly in the surgical population. Risk-factor modification and inpatient prevention strategies can have a significant impact on the incidence of HAP (Kieninger and Lipsett, 2009).
Saturday, November 26, 2011
Innate Immune Response to Pathogens and Recent Advances in Microbiology Researches
An immune system is a system of biological structures and processes within an organism that protects against disease by identifying and killing pathogens and tumor cells. It detects a wide variety of agents, from viruses to parasitic worms, and needs to distinguish them from the organism's own healthy cells and tissues in order to function properly. Detection is complicated as pathogens can evolve rapidly, and adapt to avoid the immune system and allow the pathogens to successfully infect their hosts.
To survive this challenge, multiple mechanisms evolved that recognize and neutralize pathogens. Even simple unicellular organisms such as bacteria possess enzyme systems that protect against viral infections. Other basic immune mechanisms evolved in ancient eukaryotes and remain in their modern descendants, such as plants and insects. These mechanisms include antimicrobial peptides called defensins, phagocytosis, and the complement system. Jawed vertebrates, including humans, have even more sophisticated defense mechanisms. The typical vertebrate immune system consists of many types of proteins, cells, organs, and tissues that interact in an elaborate and dynamic network. As part of this more complex immune response, the human immune system adapts over time to recognize specific pathogens more efficiently. This adaptation process is referred to as "adaptive immunity" or "acquired immunity" and creates immunological memory. The following chapters will discuss various mechanisms of immune response to pathogens based on laboratory evidence studies.
To survive this challenge, multiple mechanisms evolved that recognize and neutralize pathogens. Even simple unicellular organisms such as bacteria possess enzyme systems that protect against viral infections. Other basic immune mechanisms evolved in ancient eukaryotes and remain in their modern descendants, such as plants and insects. These mechanisms include antimicrobial peptides called defensins, phagocytosis, and the complement system. Jawed vertebrates, including humans, have even more sophisticated defense mechanisms. The typical vertebrate immune system consists of many types of proteins, cells, organs, and tissues that interact in an elaborate and dynamic network. As part of this more complex immune response, the human immune system adapts over time to recognize specific pathogens more efficiently. This adaptation process is referred to as "adaptive immunity" or "acquired immunity" and creates immunological memory. The following chapters will discuss various mechanisms of immune response to pathogens based on laboratory evidence studies.
Friday, November 25, 2011
Qualitative and quantitative detection of endotoxin in serum
Endotoxins are large (molecular weight, 200,000 to 1,000,000), heat-stable lipopolysaccharides (LPS) which are the major components of the cell wall of the gram-negative bacterium.There are more than 20 assays for the detection of endotoxin (McCabe, W. R., 1980), of which three have been used for the detection of endotoxin in clinical specimens: the rabbit pyrogen assay, the LAL
bioassay, and immunoassays. The method of choice would appear to be the LAL assay. The advantages of this assay are increased sensitivity, potential for quantitation, reactivity with the biologically active component lipid A, and relative convenience of operation.
The LAL Assay
In 1956, Bang (Bang, 1956) discovered that the endotoxin of a Vibrio species from seawater, pathogenic for the horseshoe crab (Limulus polyphemus), caused fatal intravascular coagulation and that endotoxin induced activation of this process in vitro. Levin, Bang, and coworkers subsequently showed that this coagulation was the result of an endotoxin-initiated reaction causing the enzymatic conversion of a clottable protein derived from the circulating blood cell (amebocyte) of the crab (Levin, et al.,1968; Young, et al., 1972). They recognized the potential for this biological reagent as a diagnostic tool and characterized its properties. A lysate from the amebocyte is extremely sensitive to the presence of endotoxin.
Coagulation system of L. polyphemus.
The coagulation system of L. polyphemus consists of several enzymes which are arranged in three pathways in a fashion which resembles the classic, alternate, and common mammalian coagulation cascade pathways, the components of which activate each other in a ‘‘cascade’’ sequence. The coagulation system of the Japanese horseshoe crab, T. tridentatus, which is considered homologous to the L. polyphemus American horseshoe crab, has been studied extensively (Fig.3) (Iwanaga, S., 1993; Iwanaga, et al., 1985). This
cascade sequence results in an amplification of the original stimulus which accounts for the sensitivity of the Limulus coagulation system to endotoxin at picogram-per-milliliter (10-12 g/ml) concentrations. An additional component of Limulus amebocytes is an anti-LPS factor which has anti-endotoxin properties (Warren, et al., 1992).
Gel clot LAL assay.
In the original version of the gel clot test, the endotoxin-activated clotting enzyme cleaves the coagulogen to form a clot. To perform this test, a small amount of LAL solution is added to an equal volume of a sample or a standard dilution in a small test tube. If, after an appropriate incubation time, a firm gel clot is formed, the test is scored positive. A firm gel clot is one that remains solid in the bottom of the reaction tube when the tube is inverted. Methods to
enhance the visualization of clot formation in microtiter volumes have been described (Gardi, et al, 1980; Hussaini, et al., 1987; Prior, et al., 1979). With all gel clotbased techniques, a semiquantitative result can be obtained through serial dilution of samples and standards.
Coagulogen-based LAL assay.
The limitations of the gel clot LAL test are the subjective endpoint and the relative lack of sensitivity. To overcome these limitations, various methods to quantitate the progress of the reaction leading to coagulogen conversion have been employed, for example, through monitoring the increase in turbidity (Dubczak, et al., 1979; Urbaschek, et al., 1985), the loss of coagulogen as the clot forms (Baek, 1983; Zhang, et al., 1988), the increase in precipitated protein (Nandan, et al., 1977; Nandan, et al, 1977), or the appearance of a peptide cleavage fragment of coagulogen (Zhang, et al., 1994).
Chromogenic LAL assay.
In the chromogenic LAL assay method (Iwanga, et al ., 1978), the coagulogen is completely or partially removed to be replaced by a chromogenic substrate (Scully, et al., 1980), a small synthetic peptide linked to a chromophore (para-nitroaniline) containing an amino acid sequence similar to that present at the site in the clotting protein cleaved by the clotting enzyme (X-Y-Gly-Arg-pNA). The chromogenic LAL assay usually has two stages: a LAL activation stage and, following the addition of the chromogenic substrate to the reaction mixture, a chromophore release stage. Release of the chromophore imparts a yellow color to the solution. The strength of the yellow color (as measured by optical density [OD] at 405 nm in a spectrophotometer) is a function of the amount of active clotting enzyme (and indirectly to the amount of endotoxin)
present in the solution. Both phases of the chromogenic reaction are critically time and temperature dependent, but within these limitations the chromogenic assay is sensitive to 10 pg/ml (Thomas, et al., 1981). A single-step chromogenic assay has been described (Duner, K. I., 1993; Lindsay, et al., 1989).
Specificity of the LAL Assay
The two pathways leading to the coagulation of LAL, one activated by endotoxin triggered by factor C and the other activated by b-glucans triggered by a glucan-reactive factor G, can be specifically blocked by polymyxin and laminarin, respectively (Zhang, et al., 1994). Hence, reactivity with the LAL assay that is inhibited by polymyxin B can be used as specific evidence for endotoxin. LAL derived from the Japanese horseshoe crab and from which this factor G has been removed has been promoted as an endotoxin-specific reagent (Obayashi, et al., 1985, Obayashi, et al., 1986).
LAL Endotoxin Assay for Blood Samples
When the LAL assay is used to detect endotoxin in blood, two obstacles are encountered: (i) the complex and poorly understood inhibitory factors and (ii) the levels of endotoxemia generally being at the limit of test detection. Schematic overview 1. illustrates the complex interaction among components of blood, endotoxin, and LAL. Endotoxin interacts with several components of plasma, including bile salts, proteins, and lipoproteins, leading to disaggregation, some inactivation, and the formation of complexes. These multiple effects of plasma on the activity of endotoxin are not always apparent as inactivation. (Beller, et al., 1963).
Inhibition by plasma and serum.
The loss of reactivity to LAL on addition of endotoxin to plasma or serum is partly reversible, in that reactivity can be restored by dilution with distilled water or saline (Levin, et al., 1970), and partly irreversible (Johnson, et al., 1977). The ability of plasma or serum to inhibit endotoxin activity is time dependent and temperature sensitive, being maximal at 37 to 45°C and abolished after plasma or serum is heated at 60°C for 5 min, and varies in proportion to the endotoxin potency. These characteristics imply an enzymatic inactivation of endotoxin by native plasma (Johnson, et al., 1977; Novitsky, et al., 1985, Novitsky, et al., 1985, Obayashi, T., 1984, Olofsson, et al., 1986, Webster, et al., 1980), although this has yet to be definitively demonstrated.
Endotoxemia without Sepsis
Liver disease. Endotoxemia has been suspected of having pathogenic properties in patients with liver disease even in the absence of overt gram-negative sepsis (Nolan, J. P., 1975). The origin of endotoxin in this setting is also believed to be from the gastrointestinal tract because several studies have found a portal-to-systemic gradient of endotoxin level, with higher level in portal venous blood than in peripheral blood ( Bigatello, et al, 1987; Jacob, et al, 1977; Lumsden, et al, 1988; Prytz, et al, 1976).
Hemodialysis. pyrogenic reactions are an important problemwith hemodialysis, and there is concern that this is due to contamination of the dialysis water with bacteria or endotoxin (Pegues, et al, 1992; Raij, et al, 1973) or contamination resulting from the use of reprocessed dialyzers (Flaherty, et al, 1993; Gordon, et al, 1988). There is uncertainty as to whether endotoxin is able to cross the different types of dialyzer membranes and also whether the LAL-
reactive material (LAL-RM) found in the plasma of patients undergoing hemodialysis is something other than endotoxin. It is suggested that the LAL-RM is a cellulose-based material, possibly (1-3)-β-D-glucans, which has properties distinct from endotoxin (Roslansky, et al, 1991) and reacts with the factor G-drive pathway of LAL (Zhang, et al,1994). An endotoxin specific assay which does not react with (1-3)- β-D-glucans has been developed and applied (Taniguchi, et al, 1990). In any event, LAL testing of plasma of hemodialysis patients has limited ability to detect pyrogenic reactions, having positive and negative predictive values of less than 70% (Gordon, et al, 1992).
Intestinal endotoxemia. An origin from the gastrointestinal tract has often been presumed for endotoxemia in patients with gastrointestinal diseases (Cooperstock, et al, 1985; Wellmann, et al, 1984) and also in patients receiving radiotherapy to the abdomen in association with symptoms of nausea (Maxwell, et al, 1986).
Other conditions. Transient endotoxemia occurs in patients undergoing minimally invasive procedures of the urinary (Garibaldi, et al, 1973; Robinson, et al, 1975; Tanaka, et al, 1988), biliary (Lumsden, et al, 1989), or gastrointestinal (Kelley, et al, 1985) tract. In general, the severity of symptoms and the degree or frequency of detection of endotoxemia in these patients are higher when gram-negative bacteria are found at the sites of these procedures. In premature neonates, there is an association between endotoxin in cord blood and growth of gram-negative bacteria from placental samples (Scheifele, et al, 1984).
bioassay, and immunoassays. The method of choice would appear to be the LAL assay. The advantages of this assay are increased sensitivity, potential for quantitation, reactivity with the biologically active component lipid A, and relative convenience of operation.
The LAL Assay
In 1956, Bang (Bang, 1956) discovered that the endotoxin of a Vibrio species from seawater, pathogenic for the horseshoe crab (Limulus polyphemus), caused fatal intravascular coagulation and that endotoxin induced activation of this process in vitro. Levin, Bang, and coworkers subsequently showed that this coagulation was the result of an endotoxin-initiated reaction causing the enzymatic conversion of a clottable protein derived from the circulating blood cell (amebocyte) of the crab (Levin, et al.,1968; Young, et al., 1972). They recognized the potential for this biological reagent as a diagnostic tool and characterized its properties. A lysate from the amebocyte is extremely sensitive to the presence of endotoxin.
Coagulation system of L. polyphemus.
The coagulation system of L. polyphemus consists of several enzymes which are arranged in three pathways in a fashion which resembles the classic, alternate, and common mammalian coagulation cascade pathways, the components of which activate each other in a ‘‘cascade’’ sequence. The coagulation system of the Japanese horseshoe crab, T. tridentatus, which is considered homologous to the L. polyphemus American horseshoe crab, has been studied extensively (Fig.3) (Iwanaga, S., 1993; Iwanaga, et al., 1985). This
cascade sequence results in an amplification of the original stimulus which accounts for the sensitivity of the Limulus coagulation system to endotoxin at picogram-per-milliliter (10-12 g/ml) concentrations. An additional component of Limulus amebocytes is an anti-LPS factor which has anti-endotoxin properties (Warren, et al., 1992).
Gel clot LAL assay.
In the original version of the gel clot test, the endotoxin-activated clotting enzyme cleaves the coagulogen to form a clot. To perform this test, a small amount of LAL solution is added to an equal volume of a sample or a standard dilution in a small test tube. If, after an appropriate incubation time, a firm gel clot is formed, the test is scored positive. A firm gel clot is one that remains solid in the bottom of the reaction tube when the tube is inverted. Methods to
enhance the visualization of clot formation in microtiter volumes have been described (Gardi, et al, 1980; Hussaini, et al., 1987; Prior, et al., 1979). With all gel clotbased techniques, a semiquantitative result can be obtained through serial dilution of samples and standards.
Coagulogen-based LAL assay.
The limitations of the gel clot LAL test are the subjective endpoint and the relative lack of sensitivity. To overcome these limitations, various methods to quantitate the progress of the reaction leading to coagulogen conversion have been employed, for example, through monitoring the increase in turbidity (Dubczak, et al., 1979; Urbaschek, et al., 1985), the loss of coagulogen as the clot forms (Baek, 1983; Zhang, et al., 1988), the increase in precipitated protein (Nandan, et al., 1977; Nandan, et al, 1977), or the appearance of a peptide cleavage fragment of coagulogen (Zhang, et al., 1994).
Chromogenic LAL assay.
In the chromogenic LAL assay method (Iwanga, et al ., 1978), the coagulogen is completely or partially removed to be replaced by a chromogenic substrate (Scully, et al., 1980), a small synthetic peptide linked to a chromophore (para-nitroaniline) containing an amino acid sequence similar to that present at the site in the clotting protein cleaved by the clotting enzyme (X-Y-Gly-Arg-pNA). The chromogenic LAL assay usually has two stages: a LAL activation stage and, following the addition of the chromogenic substrate to the reaction mixture, a chromophore release stage. Release of the chromophore imparts a yellow color to the solution. The strength of the yellow color (as measured by optical density [OD] at 405 nm in a spectrophotometer) is a function of the amount of active clotting enzyme (and indirectly to the amount of endotoxin)
present in the solution. Both phases of the chromogenic reaction are critically time and temperature dependent, but within these limitations the chromogenic assay is sensitive to 10 pg/ml (Thomas, et al., 1981). A single-step chromogenic assay has been described (Duner, K. I., 1993; Lindsay, et al., 1989).
Specificity of the LAL Assay
The two pathways leading to the coagulation of LAL, one activated by endotoxin triggered by factor C and the other activated by b-glucans triggered by a glucan-reactive factor G, can be specifically blocked by polymyxin and laminarin, respectively (Zhang, et al., 1994). Hence, reactivity with the LAL assay that is inhibited by polymyxin B can be used as specific evidence for endotoxin. LAL derived from the Japanese horseshoe crab and from which this factor G has been removed has been promoted as an endotoxin-specific reagent (Obayashi, et al., 1985, Obayashi, et al., 1986).
LAL Endotoxin Assay for Blood Samples
When the LAL assay is used to detect endotoxin in blood, two obstacles are encountered: (i) the complex and poorly understood inhibitory factors and (ii) the levels of endotoxemia generally being at the limit of test detection. Schematic overview 1. illustrates the complex interaction among components of blood, endotoxin, and LAL. Endotoxin interacts with several components of plasma, including bile salts, proteins, and lipoproteins, leading to disaggregation, some inactivation, and the formation of complexes. These multiple effects of plasma on the activity of endotoxin are not always apparent as inactivation. (Beller, et al., 1963).
Inhibition by plasma and serum.
The loss of reactivity to LAL on addition of endotoxin to plasma or serum is partly reversible, in that reactivity can be restored by dilution with distilled water or saline (Levin, et al., 1970), and partly irreversible (Johnson, et al., 1977). The ability of plasma or serum to inhibit endotoxin activity is time dependent and temperature sensitive, being maximal at 37 to 45°C and abolished after plasma or serum is heated at 60°C for 5 min, and varies in proportion to the endotoxin potency. These characteristics imply an enzymatic inactivation of endotoxin by native plasma (Johnson, et al., 1977; Novitsky, et al., 1985, Novitsky, et al., 1985, Obayashi, T., 1984, Olofsson, et al., 1986, Webster, et al., 1980), although this has yet to be definitively demonstrated.
Endotoxemia without Sepsis
Liver disease. Endotoxemia has been suspected of having pathogenic properties in patients with liver disease even in the absence of overt gram-negative sepsis (Nolan, J. P., 1975). The origin of endotoxin in this setting is also believed to be from the gastrointestinal tract because several studies have found a portal-to-systemic gradient of endotoxin level, with higher level in portal venous blood than in peripheral blood ( Bigatello, et al, 1987; Jacob, et al, 1977; Lumsden, et al, 1988; Prytz, et al, 1976).
Hemodialysis. pyrogenic reactions are an important problemwith hemodialysis, and there is concern that this is due to contamination of the dialysis water with bacteria or endotoxin (Pegues, et al, 1992; Raij, et al, 1973) or contamination resulting from the use of reprocessed dialyzers (Flaherty, et al, 1993; Gordon, et al, 1988). There is uncertainty as to whether endotoxin is able to cross the different types of dialyzer membranes and also whether the LAL-
reactive material (LAL-RM) found in the plasma of patients undergoing hemodialysis is something other than endotoxin. It is suggested that the LAL-RM is a cellulose-based material, possibly (1-3)-β-D-glucans, which has properties distinct from endotoxin (Roslansky, et al, 1991) and reacts with the factor G-drive pathway of LAL (Zhang, et al,1994). An endotoxin specific assay which does not react with (1-3)- β-D-glucans has been developed and applied (Taniguchi, et al, 1990). In any event, LAL testing of plasma of hemodialysis patients has limited ability to detect pyrogenic reactions, having positive and negative predictive values of less than 70% (Gordon, et al, 1992).
Intestinal endotoxemia. An origin from the gastrointestinal tract has often been presumed for endotoxemia in patients with gastrointestinal diseases (Cooperstock, et al, 1985; Wellmann, et al, 1984) and also in patients receiving radiotherapy to the abdomen in association with symptoms of nausea (Maxwell, et al, 1986).
Other conditions. Transient endotoxemia occurs in patients undergoing minimally invasive procedures of the urinary (Garibaldi, et al, 1973; Robinson, et al, 1975; Tanaka, et al, 1988), biliary (Lumsden, et al, 1989), or gastrointestinal (Kelley, et al, 1985) tract. In general, the severity of symptoms and the degree or frequency of detection of endotoxemia in these patients are higher when gram-negative bacteria are found at the sites of these procedures. In premature neonates, there is an association between endotoxin in cord blood and growth of gram-negative bacteria from placental samples (Scheifele, et al, 1984).
Thursday, November 24, 2011
Cellular and Humoral Response Involved in Gram-Negative Bacterial Sepsis and Septic Shock
As soon as a bacterium enters the body, it is confronted with two lines of defense: a humoral line and a cellular line. The humoral factors comprise complement, antibodies, and acute phase proteins. In the cellular line of defense, in particular the mononuclear cells (monocytes and macrophages) and the neutrophils are of great significance since these cells may recognize bacterial cell wall constituents directly or indirectly after complement and antibody bind to the bacterium and its constituents. It is now thought that continuous challenges with small amounts of bacterial constituents may be necessary to keep the immune system alert to infections. Indeed, low levels of LPS are present in healthy individuals without causing disease (Takakuwa, et al., 1994; Vogel, et al., 1990).
Lipopolysaccharide
While the terms endotoxin and LPS are used interchangeably, the former term to emphasize the biological activity and the latter term to refer particularly to the chemical structure and composition of the molecule (Hitchcock, et al., 1986; Qureshi, et al., 1991). LPS is a major constituent of the outer membrane of gram-negative bacteria and is the only lipid constituent of the outer leaflet (Rietschel, et al., 1994). LPS is an essential compound of the cell wall and is a
prerequisite for bacterial viability. It is not a toxic molecule when it is incorporated into the bacterial outer membrane, but after release from the bacterial wall, its toxic moiety, lipid A, is exposed to immune cells, thus evoking an inflammatory response. LPS and other cell wall constituents are released from the bacterial cells when they multiply but also when bacteria die or lyse (Hellman, et al., 2000, Rietschel, et al., 1994). Various endogenous factors like complement and bactericidal proteins can cause disintegration of bacteria, resulting in the release of LPS (De Bleser, et al., 1994). In addition, some antibiotics are known to cause the release of LPS from bacteria (Crosby, et al., 1994).
The LPS molecule consists of four different parts (Fig1,2). (Lugtenberg, et al., 1983; Raetz, et al., 1990; Rietschel, et al., 1994).The first and most essential part is lipid A, the covalently linked lipid component of LPS. Six or more fatty acid residues are linked to two phosphorylated glucosamine sugars. All bacterial species carry unique LPS. Experiments with synthetic lipid A have shown that this part of the LPS molecule represents the toxic moiety (Kotani, et al 1985). The second part of the LPS molecule is the inner core, which consists of two or more 2-keto-3-deoxyoctonic acid (KDO) sugars linked to the lipid A glucosamine and two or three heptose (L-glycero-D-manno-heptose) sugars linked to the KDO. Both sugars are unique to bacteria. The outer core, the third part of the LPS molecule, consists of common sugars and is more variable than the inner core. It is normally three sugars long with one or more covalently bound sugars as side chains. LPS serotypes consisting of lipid A and the complete inner and outer core are denoted Ra-LPS, whereas the Rb- and Rc-LPS serotypes only contain a part of the outer core. The fourth moiety of the LPS molecule is the O antigen. This part of the LPS molecule is attached to the
terminal sugar of the outer core, extends from the bacterial surface, and is highly immunogenic. It is composed of units of common sugars, but there is a huge interspecies and interstrain variation in the composition and length (Edwin S, et al., 2003).
Cellular defense:
LPS and other bacterial (surface) components are recognized by complement and antibodies, leading to opsonisation and lysis of the bacterium. Phagocytes (monocytes, macrophages, and polymorphonuclear leukocytes [PMN]) are able to recognize opsonized bacterial components by complement receptors and Fc receptors (which bind immunoglobulin G [IgG] antibodies) (Frank, et al., 1991).
In the host response to bacteria, the mononuclear phagocytes (monocytes and macrophages) are of major importance. Recognition of LPS or other bacterial components by these cells initiates a cascade of release of inflammatory mediators, vascular and physiological changes, and recruitment of immune cells. An LPS-activated macrophage becomes metabolically active and produces intracellular stores of oxygen free radicals and other microbicidal agents (lysozyme, cationic proteins, acid hydrolases, and lactoferrin) and secretes inflammatory mediators (Hiemstra, et al., 1993; Mayer, et al., 1991; Roitt, I. M., 1994). One of the key mediators is TNF-α which is one of the first cytokines released by macrophages (Beutler, et al., 1985). The release of TNF-α,
IL-1, IL-6, IL-8, IL-12, platelet-activating factor (PAF), chemokines, and eicosanoids has profound effects on the surrounding tissue (Hack, et al., 1997; Katori, et al., 2000; Lukacs, et al., 1999).
The extravasation of PMN is enabled by vasodilatation and upregulation of adhesion molecules on endothelial cells, PMN, and macrophages (Jaeschke, H., and C. W. Smith., 1997; Kawamura, et al., 1995; Van Oosten, et al., 1995). The PMN react to these stimuli by intravascular aggregation, adherence to the endothelium, diapedesis, and the production of inflammatory mediators like TNF-α, leukotriene B4, and PAF (Mulligan, et al., 1993; Van Epps, et al., 1993). The (activated) PMN express CD14, CD11/CD18, and several complement and Fc receptors and are thus able to recognize and phagocytose LPS, bacterial fragments, and whole bacteria. As specialized phagocytes, PMN produce an impressive series of microbicidal agents, such as lysozyme, bactericidal/permeability increasing protein (BPI), enzymes, and oxygen free radicals (Chatham, et al., 1993; Roitt, I. M., 1994). These agents are used mainly for lysosomal killing of microorganisms. However, adherence of the PMN to endothelial cells and the presence of high concentrations of stimuli may also result in the release of microbicidal agents; much of the endothelial damage observed in sepsis is caused by these agents (Bone, et al., 1991). Endothelial cells respond to LPS (via soluble CD14) and to the circulating cytokines by the release of IL-1, IL-6, eicosanoids, the vasoactive agents endothelium derived relaxation factor, endothelin-1, chemokines, and colony stimulating factors (CSF) (Mahalingam, et al., 1999).
The inflammatory mediators secreted by the different cell populations attract and activate B and T lymphocytes. In turn, the latter release mediators such as
IL-2, gamma interferon (IFN-γ), and granulocyte-macrophage (GM)-CSF. IL-2 and GM-CSF are involved in proliferation and activation of PMN and mononuclear cells, whereas IFN-γ enhances the effects of LPS on mononuclear cells (Bone, R. C., 1991; Heinzel, et al., 1994; Jaeschke, H., 1996; Ying, et al.,1993). The actions of the activated immune cells combined with the effects of the inflammatory mediators cause symptoms such as fever, endothelial damage, capillary leakage, peripheral vascular dilatation, coagulation disorders, microthrombi, and myocardial depression. These phenomena may finally result in multiple organ dysfunction, shock, and death (Bone, et al., 1991).
Humeral response
Bacteria activate both complement pathways: i) alternative pathway which is triggered by binding polysaccharide surface components (O antigen, capsule, and LPS) to complement factor 3 (C3) (Joiner, et al., 1984; Quezado, et al., 1994; Tesh, et al., 1988) ii) classical pathway which is activated by binding Lipid A to C1q (Ying, et al., 1993). The classical complement pathway is also activated in the presence of specific antibodies (IgG and IgM) against gram-negative bacterial constituents. In all three cases, C3b is deposited on the molecule or cell surface, which promotes phagocytosis by macrophages and neutrophils and leads to insertion of C5–C9 (membrane attack complex) into the cell surface, leading to lysis of the bacterium (De Boer, et al., 1993; Frank, et al., 1991). However, long O-antigen chains in gram-negative bacteria may protect the bacteria from complement-mediated lysis (Haeney, M. R., 1998). With the cleavage of C3 and C5, the chemoattractive and vasoactive agents C3a and C5a are released. They cause increased vascular permeability, upregulate adhesion molecule expression on endothelial cells and neutrophils, and attract and activate
these phagocytes. Furthermore, they activate basophilic granulocytes and mast cells: these cells release a variety of vasoactive compounds (such ashistamine), facilitating the invasion of phagocytes. (Espevik, et al., 1993; Hsueh, et al., 1990; Kuipers, et al., 1994; Mulligan, et al., 1993; Pu¨schel, et al., 1993; Roitt, I. M., 1994; Tesh, et al., 1988; Van Epps, et al., 1993)
During infection, liver parenchymal cells are stimulated by TNF-α, IL-1, and IL-6 to produce acute-phase proteins. These proteins comprise C-reactive protein, serum amyloid A, lipopolysaccharide- binding protein (LBP), serum amyloid P, hemopexin, haptoglobin, complement C3 and C9, α1-acid glycoprotein, α2-macroglobulin, and some proteinase inhibitors (476, 498). The expression is differentially upregulated from several fold (C3 and C9) to even 1,000-fold (C-reactive protein). Some of the acute-phase proteins, like LBP modulate the immune response reactions by activation of phagocytes and antigen-presenting cells, but basically the acute-phase response is considered to alleviate the damage caused during infection (Fey, et al., 1994; Kuipers, et al., 1994; Ramadori, et al., 1990). Albumin is a so-called negative acute-phase protein since its production is down regulated during inflammation (Fey, et al., 1994).
Lipopolysaccharide
While the terms endotoxin and LPS are used interchangeably, the former term to emphasize the biological activity and the latter term to refer particularly to the chemical structure and composition of the molecule (Hitchcock, et al., 1986; Qureshi, et al., 1991). LPS is a major constituent of the outer membrane of gram-negative bacteria and is the only lipid constituent of the outer leaflet (Rietschel, et al., 1994). LPS is an essential compound of the cell wall and is a
prerequisite for bacterial viability. It is not a toxic molecule when it is incorporated into the bacterial outer membrane, but after release from the bacterial wall, its toxic moiety, lipid A, is exposed to immune cells, thus evoking an inflammatory response. LPS and other cell wall constituents are released from the bacterial cells when they multiply but also when bacteria die or lyse (Hellman, et al., 2000, Rietschel, et al., 1994). Various endogenous factors like complement and bactericidal proteins can cause disintegration of bacteria, resulting in the release of LPS (De Bleser, et al., 1994). In addition, some antibiotics are known to cause the release of LPS from bacteria (Crosby, et al., 1994).
The LPS molecule consists of four different parts (Fig1,2). (Lugtenberg, et al., 1983; Raetz, et al., 1990; Rietschel, et al., 1994).The first and most essential part is lipid A, the covalently linked lipid component of LPS. Six or more fatty acid residues are linked to two phosphorylated glucosamine sugars. All bacterial species carry unique LPS. Experiments with synthetic lipid A have shown that this part of the LPS molecule represents the toxic moiety (Kotani, et al 1985). The second part of the LPS molecule is the inner core, which consists of two or more 2-keto-3-deoxyoctonic acid (KDO) sugars linked to the lipid A glucosamine and two or three heptose (L-glycero-D-manno-heptose) sugars linked to the KDO. Both sugars are unique to bacteria. The outer core, the third part of the LPS molecule, consists of common sugars and is more variable than the inner core. It is normally three sugars long with one or more covalently bound sugars as side chains. LPS serotypes consisting of lipid A and the complete inner and outer core are denoted Ra-LPS, whereas the Rb- and Rc-LPS serotypes only contain a part of the outer core. The fourth moiety of the LPS molecule is the O antigen. This part of the LPS molecule is attached to the
terminal sugar of the outer core, extends from the bacterial surface, and is highly immunogenic. It is composed of units of common sugars, but there is a huge interspecies and interstrain variation in the composition and length (Edwin S, et al., 2003).
Cellular defense:
LPS and other bacterial (surface) components are recognized by complement and antibodies, leading to opsonisation and lysis of the bacterium. Phagocytes (monocytes, macrophages, and polymorphonuclear leukocytes [PMN]) are able to recognize opsonized bacterial components by complement receptors and Fc receptors (which bind immunoglobulin G [IgG] antibodies) (Frank, et al., 1991).
In the host response to bacteria, the mononuclear phagocytes (monocytes and macrophages) are of major importance. Recognition of LPS or other bacterial components by these cells initiates a cascade of release of inflammatory mediators, vascular and physiological changes, and recruitment of immune cells. An LPS-activated macrophage becomes metabolically active and produces intracellular stores of oxygen free radicals and other microbicidal agents (lysozyme, cationic proteins, acid hydrolases, and lactoferrin) and secretes inflammatory mediators (Hiemstra, et al., 1993; Mayer, et al., 1991; Roitt, I. M., 1994). One of the key mediators is TNF-α which is one of the first cytokines released by macrophages (Beutler, et al., 1985). The release of TNF-α,
IL-1, IL-6, IL-8, IL-12, platelet-activating factor (PAF), chemokines, and eicosanoids has profound effects on the surrounding tissue (Hack, et al., 1997; Katori, et al., 2000; Lukacs, et al., 1999).
The extravasation of PMN is enabled by vasodilatation and upregulation of adhesion molecules on endothelial cells, PMN, and macrophages (Jaeschke, H., and C. W. Smith., 1997; Kawamura, et al., 1995; Van Oosten, et al., 1995). The PMN react to these stimuli by intravascular aggregation, adherence to the endothelium, diapedesis, and the production of inflammatory mediators like TNF-α, leukotriene B4, and PAF (Mulligan, et al., 1993; Van Epps, et al., 1993). The (activated) PMN express CD14, CD11/CD18, and several complement and Fc receptors and are thus able to recognize and phagocytose LPS, bacterial fragments, and whole bacteria. As specialized phagocytes, PMN produce an impressive series of microbicidal agents, such as lysozyme, bactericidal/permeability increasing protein (BPI), enzymes, and oxygen free radicals (Chatham, et al., 1993; Roitt, I. M., 1994). These agents are used mainly for lysosomal killing of microorganisms. However, adherence of the PMN to endothelial cells and the presence of high concentrations of stimuli may also result in the release of microbicidal agents; much of the endothelial damage observed in sepsis is caused by these agents (Bone, et al., 1991). Endothelial cells respond to LPS (via soluble CD14) and to the circulating cytokines by the release of IL-1, IL-6, eicosanoids, the vasoactive agents endothelium derived relaxation factor, endothelin-1, chemokines, and colony stimulating factors (CSF) (Mahalingam, et al., 1999).
The inflammatory mediators secreted by the different cell populations attract and activate B and T lymphocytes. In turn, the latter release mediators such as
IL-2, gamma interferon (IFN-γ), and granulocyte-macrophage (GM)-CSF. IL-2 and GM-CSF are involved in proliferation and activation of PMN and mononuclear cells, whereas IFN-γ enhances the effects of LPS on mononuclear cells (Bone, R. C., 1991; Heinzel, et al., 1994; Jaeschke, H., 1996; Ying, et al.,1993). The actions of the activated immune cells combined with the effects of the inflammatory mediators cause symptoms such as fever, endothelial damage, capillary leakage, peripheral vascular dilatation, coagulation disorders, microthrombi, and myocardial depression. These phenomena may finally result in multiple organ dysfunction, shock, and death (Bone, et al., 1991).
Humeral response
Bacteria activate both complement pathways: i) alternative pathway which is triggered by binding polysaccharide surface components (O antigen, capsule, and LPS) to complement factor 3 (C3) (Joiner, et al., 1984; Quezado, et al., 1994; Tesh, et al., 1988) ii) classical pathway which is activated by binding Lipid A to C1q (Ying, et al., 1993). The classical complement pathway is also activated in the presence of specific antibodies (IgG and IgM) against gram-negative bacterial constituents. In all three cases, C3b is deposited on the molecule or cell surface, which promotes phagocytosis by macrophages and neutrophils and leads to insertion of C5–C9 (membrane attack complex) into the cell surface, leading to lysis of the bacterium (De Boer, et al., 1993; Frank, et al., 1991). However, long O-antigen chains in gram-negative bacteria may protect the bacteria from complement-mediated lysis (Haeney, M. R., 1998). With the cleavage of C3 and C5, the chemoattractive and vasoactive agents C3a and C5a are released. They cause increased vascular permeability, upregulate adhesion molecule expression on endothelial cells and neutrophils, and attract and activate
these phagocytes. Furthermore, they activate basophilic granulocytes and mast cells: these cells release a variety of vasoactive compounds (such ashistamine), facilitating the invasion of phagocytes. (Espevik, et al., 1993; Hsueh, et al., 1990; Kuipers, et al., 1994; Mulligan, et al., 1993; Pu¨schel, et al., 1993; Roitt, I. M., 1994; Tesh, et al., 1988; Van Epps, et al., 1993)
During infection, liver parenchymal cells are stimulated by TNF-α, IL-1, and IL-6 to produce acute-phase proteins. These proteins comprise C-reactive protein, serum amyloid A, lipopolysaccharide- binding protein (LBP), serum amyloid P, hemopexin, haptoglobin, complement C3 and C9, α1-acid glycoprotein, α2-macroglobulin, and some proteinase inhibitors (476, 498). The expression is differentially upregulated from several fold (C3 and C9) to even 1,000-fold (C-reactive protein). Some of the acute-phase proteins, like LBP modulate the immune response reactions by activation of phagocytes and antigen-presenting cells, but basically the acute-phase response is considered to alleviate the damage caused during infection (Fey, et al., 1994; Kuipers, et al., 1994; Ramadori, et al., 1990). Albumin is a so-called negative acute-phase protein since its production is down regulated during inflammation (Fey, et al., 1994).
Tuesday, November 22, 2011
Nucleic Acid Analysis Without Amplification
It is molecular methods not based on the amplification of the target have been used.
Nucleic acid analysis without Amplification (Nucleic acid probe technology)
A nucleic acid probe is a labeled sequence of single stranded DNA or RNA that can hybridise specifically with its complementary sequence (Smith, 2002).
Nucleic acid hybridization technique
Fluorescent in situ hybridization (FISH) is a tool that today is widely used for identification, visualization and localization of microorganisms in many fields of microbiology
FISH was mainly applied in connection with environmental samples during the first years but it became clear that the method also has advantages in diagnostic microbiology for rapid identification and direct visualization of bacteria
Nucleic hybridization refer to formation of hydrogen bonds between nucleotides of single stranded DNA and/or RNA molecules that are complementary to each other. This form a stable double stranded nucleic acid molecule. The resulting double stranded hybrids may be DNA:DNA; DNA:RNA, or RNA:RNA.
This hybridization process called duplex formation. This process is key of component for many testes including blotting methods, PCR, and other molecular based techniques.
The two single stranded nucleic acid molecules used in hybridization techniques are referred to by different terms. One of the strands is known as the target.
The target strand is the DNA or RNA sequence that will be identified by the employed molecular diagnostics method. The target is referred to as the template it can be either immobilized on a solid support mechanism or is suspended in solution.The other strand is called the probe. The probe is usually a single stranded DNA or RNA oligonucleotide that is labeled with an attached reporter chemical or a radionucleotide that can be detected either visually, by film, or by an instrument. The probe is produced synthetically to detect a specific target.
Hybridization reaction variables
Several variables affect the outcome of a given hybridization reaction. These variable include;
-Temperature
The stability of a given hybrid can be calculated by determining the melting temperature (™) of a probe. The Tm is the temperature at which 50% of hybrids have formed and 50% of the single stranded nucleic acid molecules are still dissociated. Tm is dependent on the G+C ratio because three hydrogen bonds form between G and C, instead of the two hydrogen bonds that form between adenine (A) and (T);The G\C bond pair is more thermodynamically stable than the A\T bond pair.
-Length of the probe
Another aspect that affects the Tm is the length of the probe ; in general the Tm is lower for a shorter probe.Hybridization reactions tend to occur more rapidly for shorter probes than for longer probes
-Probe Concentration
Higher probe concentrations typically lower the reaction time by saturating all of the available probe target sequences. However, excessive probe concentrations promote nonspecific binding of the probe to non target sequences
-Salt concentration
The rate of a hybridization reaction will increase as the salt concentration increases, up to a threshold; past 1.2 M Nacl, the rate of the reaction become constant.
-PH
Neutral PH is preferable for most hybridization reactions
-Probe selection
Selection of proper probe for nucleic acid hybridization reaction is important as the hybridization method itself. probes may be either DNA or RNA based, and are either radiolabeled or non isotopically labeled.
Radiolabeled probes are rarely used due to have short half lives and un desirable waste.radiolabeled probes have been replaced by nonisotopic labels, including biotin digoxigenin (DIG), and fluorescein, nonisotopic labels have resolution and sensitivity that approache .A probe may also be end labeled or continuously labeled
Hybridization Formats
Hybridization reaction may occur in a solid support mechanism, in situ or in solution
A-Solid support hybridization
Technique often called (blotting) the target nucleic acid is transferred and immobilized to a membrane, composed of either nitrocellulose or nylon. Labeled probe is then hybridized to the immobilized nucleic acid washing steps are used to remove excess probe. Two examples of solid support hybridization techniques are:
1-Southern Blot
The southern blot was first described in 1975 by E.M southern; he described a technique whereby chromosomal DNA has digested with a restriction enzyme then separated by agarose gel electrophoresis, then transferred and immobilized to a nitro cellulose membrane, then labeled probe is hybridized to the specific target DNA sequences. [Figure, 5]The southern blot takes more than one day to perform, it can be used to identified micro organism, to detect mutation, to type strains for epidemiological investigation, and for other purposes (Vanrompay.d., 2000)
2- Northern Blot
A northern blot was first described by Alwine et al 1977; northern blot used to determine the size of particular RNA transcript. It has the same procedure of the southern blot but with difference, the restriction enzyme is not used to digest RNA before separation due to RNA is small enough to be separated by agaros gel electrophoresis. Like southern blotting, northern blotting is not often used in clinical microbiology laboratories
3-In Situ Hybridization
In Situ Hybridization (ISH). This is a method of hybridization wherein DNA or RNA transcript can be detected directly in the tissue with labeled probes.ISH may used to detected low level of viruses in tissue specimen such as human papillomaviruses (HPV)
An example is fluorescence in situ hybridization (FISH) with oligonucleotide probes targeting bacterial or fungal genes (typically rRNA genes) As conventional FISH probes, they are usually designed to target naturally abundant rRNA genes, thereby allowing the detection of microorganisms without the need for an amplification step .
An evolution of classical oligonucleotide probes are peptide nucleic acid (PNA) probes, which are synthetic oligomers mimicking the DNA or the RNA structure. In PNA probes, the negatively charged (deoxy)ribose-phosphate nucleic acid backbone is replaced by an uncharged N-(2-aminoethyl)-glycine scaffold to which the nucleotide bases are attached via a methylene carbonyl linker (151). Due to their neutral charge, PNA probes have more robust hybridization characteristics than those of DNA probes. As conventional FISH probes, they are usually designed to target naturally abundant rRNA genes, thereby allowing the detection of microorganisms without the need for an amplification step
Finally, and adding to their clinical applicability, PNA-FISH probes are less susceptible to inhibition by impurities in different clinical samples than amplified NAT-based methods
B-In Solution Hybridization
In solution hybridization is the type of hybridization reaction most often used by clinical microbiology laboratories.Hybridization between a labeled probe and target nucleic acids in a liquid solution in tubes or in microtiter wells, usually detection methods are chemiluminescent based. It used to rapidly identify infectious disease organisms.
Nucleic acid analysis without Amplification (Nucleic acid probe technology)
A nucleic acid probe is a labeled sequence of single stranded DNA or RNA that can hybridise specifically with its complementary sequence (Smith, 2002).
Nucleic acid hybridization technique
Fluorescent in situ hybridization (FISH) is a tool that today is widely used for identification, visualization and localization of microorganisms in many fields of microbiology
FISH was mainly applied in connection with environmental samples during the first years but it became clear that the method also has advantages in diagnostic microbiology for rapid identification and direct visualization of bacteria
Nucleic hybridization refer to formation of hydrogen bonds between nucleotides of single stranded DNA and/or RNA molecules that are complementary to each other. This form a stable double stranded nucleic acid molecule. The resulting double stranded hybrids may be DNA:DNA; DNA:RNA, or RNA:RNA.
This hybridization process called duplex formation. This process is key of component for many testes including blotting methods, PCR, and other molecular based techniques.
The two single stranded nucleic acid molecules used in hybridization techniques are referred to by different terms. One of the strands is known as the target.
The target strand is the DNA or RNA sequence that will be identified by the employed molecular diagnostics method. The target is referred to as the template it can be either immobilized on a solid support mechanism or is suspended in solution.The other strand is called the probe. The probe is usually a single stranded DNA or RNA oligonucleotide that is labeled with an attached reporter chemical or a radionucleotide that can be detected either visually, by film, or by an instrument. The probe is produced synthetically to detect a specific target.
Hybridization reaction variables
Several variables affect the outcome of a given hybridization reaction. These variable include;
-Temperature
The stability of a given hybrid can be calculated by determining the melting temperature (™) of a probe. The Tm is the temperature at which 50% of hybrids have formed and 50% of the single stranded nucleic acid molecules are still dissociated. Tm is dependent on the G+C ratio because three hydrogen bonds form between G and C, instead of the two hydrogen bonds that form between adenine (A) and (T);The G\C bond pair is more thermodynamically stable than the A\T bond pair.
-Length of the probe
Another aspect that affects the Tm is the length of the probe ; in general the Tm is lower for a shorter probe.Hybridization reactions tend to occur more rapidly for shorter probes than for longer probes
-Probe Concentration
Higher probe concentrations typically lower the reaction time by saturating all of the available probe target sequences. However, excessive probe concentrations promote nonspecific binding of the probe to non target sequences
-Salt concentration
The rate of a hybridization reaction will increase as the salt concentration increases, up to a threshold; past 1.2 M Nacl, the rate of the reaction become constant.
-PH
Neutral PH is preferable for most hybridization reactions
-Probe selection
Selection of proper probe for nucleic acid hybridization reaction is important as the hybridization method itself. probes may be either DNA or RNA based, and are either radiolabeled or non isotopically labeled.
Radiolabeled probes are rarely used due to have short half lives and un desirable waste.radiolabeled probes have been replaced by nonisotopic labels, including biotin digoxigenin (DIG), and fluorescein, nonisotopic labels have resolution and sensitivity that approache .A probe may also be end labeled or continuously labeled
Hybridization Formats
Hybridization reaction may occur in a solid support mechanism, in situ or in solution
A-Solid support hybridization
Technique often called (blotting) the target nucleic acid is transferred and immobilized to a membrane, composed of either nitrocellulose or nylon. Labeled probe is then hybridized to the immobilized nucleic acid washing steps are used to remove excess probe. Two examples of solid support hybridization techniques are:
1-Southern Blot
The southern blot was first described in 1975 by E.M southern; he described a technique whereby chromosomal DNA has digested with a restriction enzyme then separated by agarose gel electrophoresis, then transferred and immobilized to a nitro cellulose membrane, then labeled probe is hybridized to the specific target DNA sequences. [Figure, 5]The southern blot takes more than one day to perform, it can be used to identified micro organism, to detect mutation, to type strains for epidemiological investigation, and for other purposes (Vanrompay.d., 2000)
2- Northern Blot
A northern blot was first described by Alwine et al 1977; northern blot used to determine the size of particular RNA transcript. It has the same procedure of the southern blot but with difference, the restriction enzyme is not used to digest RNA before separation due to RNA is small enough to be separated by agaros gel electrophoresis. Like southern blotting, northern blotting is not often used in clinical microbiology laboratories
3-In Situ Hybridization
In Situ Hybridization (ISH). This is a method of hybridization wherein DNA or RNA transcript can be detected directly in the tissue with labeled probes.ISH may used to detected low level of viruses in tissue specimen such as human papillomaviruses (HPV)
An example is fluorescence in situ hybridization (FISH) with oligonucleotide probes targeting bacterial or fungal genes (typically rRNA genes) As conventional FISH probes, they are usually designed to target naturally abundant rRNA genes, thereby allowing the detection of microorganisms without the need for an amplification step .
An evolution of classical oligonucleotide probes are peptide nucleic acid (PNA) probes, which are synthetic oligomers mimicking the DNA or the RNA structure. In PNA probes, the negatively charged (deoxy)ribose-phosphate nucleic acid backbone is replaced by an uncharged N-(2-aminoethyl)-glycine scaffold to which the nucleotide bases are attached via a methylene carbonyl linker (151). Due to their neutral charge, PNA probes have more robust hybridization characteristics than those of DNA probes. As conventional FISH probes, they are usually designed to target naturally abundant rRNA genes, thereby allowing the detection of microorganisms without the need for an amplification step
Finally, and adding to their clinical applicability, PNA-FISH probes are less susceptible to inhibition by impurities in different clinical samples than amplified NAT-based methods
B-In Solution Hybridization
In solution hybridization is the type of hybridization reaction most often used by clinical microbiology laboratories.Hybridization between a labeled probe and target nucleic acids in a liquid solution in tubes or in microtiter wells, usually detection methods are chemiluminescent based. It used to rapidly identify infectious disease organisms.
Monday, November 21, 2011
Ribotyping in clincal microbiology
Ribotyping involves the fingerprinting of genomic DNA restriction fragments that contain all or part of the genes coding for the 16S and 23SrRNA. Conceptually, ribotyping is similar to probing restriction fragments of chromosomal DNA with cloned probes (randomly cloned probes or probes derived from a specific coding sequence such as that of a virulence factor).
Ribotyping assays have been used to differentiate bacterial strains in different serotypes and to determine the serotype(s)most frequently involved in outbreaks . This technique is especially useful in epidemiological studies for organisms with multiple ribosomal operons, such as members of the family of Enterobacteriaceae. Ribotyping simplifies the microrestriction patterns by rendering visible only the DNA fragments containing part or all of the ribosomal genes. The technique is less helpful when the bacterial species under investigation contains only one or a few ribosomal operons. In these instances, ribotyping typically detects only one or two bands, which limits its utility for epidemiological studies. Most studies have indicated that PFGE is superior to ribotyping for analysis of common nosocomial pathogens.
Ribotyping assays have been used to differentiate bacterial strains in different serotypes and to determine the serotype(s)most frequently involved in outbreaks . This technique is especially useful in epidemiological studies for organisms with multiple ribosomal operons, such as members of the family of Enterobacteriaceae. Ribotyping simplifies the microrestriction patterns by rendering visible only the DNA fragments containing part or all of the ribosomal genes. The technique is less helpful when the bacterial species under investigation contains only one or a few ribosomal operons. In these instances, ribotyping typically detects only one or two bands, which limits its utility for epidemiological studies. Most studies have indicated that PFGE is superior to ribotyping for analysis of common nosocomial pathogens.
Saturday, November 19, 2011
Restriction Enzyme Pattern in Clinical Microbiology
Restriction endonucleases recognize specific nucleotide sequences inDNA and produce double-stranded cleavages that break the DNAinto small fragments. The number and sizes of the restrictionfragments, called restriction fragment length polymorphisms (RFLPs) , generated by digesting microbial DNA are influenced by both therecognition sequence of the enzyme and the composition of theDNA. In conventional restriction endonuclease analysis, chromosomalor plasmid DNA is extracted from microbial specimens and thendigested with endonucleases into small fragments. These fragmentsare then separated by size with use of agarose gel electrophoresis.The nucleic acid electrophoretic pattern can then be visualizedby ethidium bromide staining and examination under UV light(Lodish et al., 2004).
Restriction endonuclease analysis has the advantage of being highly reproducible, very accurate in determining the relatedness of microbial strains, and well within the technical capabilitiesof experienced laboratory technologists. However, the majorlimitation of this technique, especially for chromosomal DNA,is the difficulty of comparing the complex profiles generated,which consist of hundreds of fragments. To address this problem,pulse-field gel electrophoresis (PFGE) has been developed to enable the separation of large DNA fragments. PFGE providesa chromosomal restriction profile typically composed of 5 to20 distinct, well-resolved fragments ranging from ~10–800kilobases (kb). The relative simplicity of the RFLP profilesgenerated by PFGE facilitates application of the procedure inidentification and epidemiological survey of bacterial pathogens. Fingerprinting,which combines PFGE with Southern transfer and hybridization,has been widely used in studying the tuberculosis nosocomialoutbreak in human immunodeficiency virus (HIV)-positive populations(Goering .,2004).
Restriction endonuclease analysis has the advantage of being highly reproducible, very accurate in determining the relatedness of microbial strains, and well within the technical capabilitiesof experienced laboratory technologists. However, the majorlimitation of this technique, especially for chromosomal DNA,is the difficulty of comparing the complex profiles generated,which consist of hundreds of fragments. To address this problem,pulse-field gel electrophoresis (PFGE) has been developed to enable the separation of large DNA fragments. PFGE providesa chromosomal restriction profile typically composed of 5 to20 distinct, well-resolved fragments ranging from ~10–800kilobases (kb). The relative simplicity of the RFLP profilesgenerated by PFGE facilitates application of the procedure inidentification and epidemiological survey of bacterial pathogens. Fingerprinting,which combines PFGE with Southern transfer and hybridization,has been widely used in studying the tuberculosis nosocomialoutbreak in human immunodeficiency virus (HIV)-positive populations(Goering .,2004).
Friday, November 18, 2011
Antibiotics-producing microorganisms
There has reports mentioned that antibiotic production is a feature of several kinds of soil bacteria and fungi and may represent a survival mechanism whereby organisms can eliminate competition and colonize a niche (Jensen et al., 1997; Talaro and Talaro, 1996). Although both fungal and bacterial species are known to produce antibiotics, fungi tend to produce mostly broad-spectrum activities but more antibiotics are produced by bacteria (Salyers and Whitt, 2001).
Oskay et al. (2004) showed that actinomycetes have the capability to synthesize many different biologically active secondary metabolites such as antibiotics, herbicides, pesticides, anti-parasitic, and enzymes like cellulase and xylanase used in waste treatment. Actinomycetes are the most widely distributed groups of microorganisms in nature. They are attractive, bodacious and charming filamentous gram-positive bacteria. They make up in many cases, especially under dry alkaline conditions, a large part of the microbial population of the soil (Athalye et al., 1981; Goodfellow and Williams, 1983; Lacey, 1973 and 1997; Nakayama, 1981; Waksman, 1961). Based on several studies among bacteria, the actinomycetes are noteworthy as antibiotic producers, making three quarters of all known products, the Streptomyces are especially prolific (Lacey, 1973; Lechevalier, 1989; Locci, 1989; Saadoun and Gharaibeh, 2003; Waksman, 1961).
Actinomycetes can be isolated from soil and marine sediments. The soil actinomycetes have been important sources of antibiotics. For example, about 1% of soil actinomycetes produce streptomycin, first discovered in the 1940s, whereas daptomycin producers were discovered only after screening nearly 107 actinomycetes. Most of the antibiotics in use today are derivatives of natural products of actinomycetes and fungi (Butler and Buss, 2006; Newman and Cragg, 2007). Antibiotics produced by actinomycetes have been evolving for ~1 billion years (Baltz, 2005 and 2006), and fitness has been tested by the ability to penetrate other microbes and inhibit the target enzymes, macromolecules or macromolecular structures (Baltz, 2008).
The ability of actinomycetes to make secondary metabolites with different useful properties is widely exploited. Two thirds of the antibiotics produced by microorganisms are made by actinomycetes. In particular, genus of Streptomyces is remarkable in this aspect, representing about 80% of the actinomycete antibiotics (Borodina et al., 2005).
Microbial natural products are the origin of most of the antibiotics. The discovery of penicillin in the 1940s was followed by the discovery of a huge number of antibiotics from microbes, in particular from members of the actinomycetes and fungi. Actinomycetes have traditionally been the most prolific group in antibiotic production. Fungi are another rich source of antibiotics (Peláez, 2006).
Anupama et al. (2007) reported that actinomycetes have been isolated from different soils, plant materials, water and marine sediments (Mincer et al., 2002). At least 90% of the population among actinomycetes isolated from soils have been reported to be Streptomyces spp. Among microorganisms, actinomycetes are the important source for bioactive metabolites especially antibiotics (Bérdy, 2005).
Oskay et al. (2004) showed that actinomycetes have the capability to synthesize many different biologically active secondary metabolites such as antibiotics, herbicides, pesticides, anti-parasitic, and enzymes like cellulase and xylanase used in waste treatment. Actinomycetes are the most widely distributed groups of microorganisms in nature. They are attractive, bodacious and charming filamentous gram-positive bacteria. They make up in many cases, especially under dry alkaline conditions, a large part of the microbial population of the soil (Athalye et al., 1981; Goodfellow and Williams, 1983; Lacey, 1973 and 1997; Nakayama, 1981; Waksman, 1961). Based on several studies among bacteria, the actinomycetes are noteworthy as antibiotic producers, making three quarters of all known products, the Streptomyces are especially prolific (Lacey, 1973; Lechevalier, 1989; Locci, 1989; Saadoun and Gharaibeh, 2003; Waksman, 1961).
Actinomycetes can be isolated from soil and marine sediments. The soil actinomycetes have been important sources of antibiotics. For example, about 1% of soil actinomycetes produce streptomycin, first discovered in the 1940s, whereas daptomycin producers were discovered only after screening nearly 107 actinomycetes. Most of the antibiotics in use today are derivatives of natural products of actinomycetes and fungi (Butler and Buss, 2006; Newman and Cragg, 2007). Antibiotics produced by actinomycetes have been evolving for ~1 billion years (Baltz, 2005 and 2006), and fitness has been tested by the ability to penetrate other microbes and inhibit the target enzymes, macromolecules or macromolecular structures (Baltz, 2008).
The ability of actinomycetes to make secondary metabolites with different useful properties is widely exploited. Two thirds of the antibiotics produced by microorganisms are made by actinomycetes. In particular, genus of Streptomyces is remarkable in this aspect, representing about 80% of the actinomycete antibiotics (Borodina et al., 2005).
Microbial natural products are the origin of most of the antibiotics. The discovery of penicillin in the 1940s was followed by the discovery of a huge number of antibiotics from microbes, in particular from members of the actinomycetes and fungi. Actinomycetes have traditionally been the most prolific group in antibiotic production. Fungi are another rich source of antibiotics (Peláez, 2006).
Anupama et al. (2007) reported that actinomycetes have been isolated from different soils, plant materials, water and marine sediments (Mincer et al., 2002). At least 90% of the population among actinomycetes isolated from soils have been reported to be Streptomyces spp. Among microorganisms, actinomycetes are the important source for bioactive metabolites especially antibiotics (Bérdy, 2005).
Thursday, November 17, 2011
Plasmid analysis
Plasmids are small, self-replicating circular DNA found in many bacteria.These often encode genes related to antibiotic resistance and certain virulence factors. In epidemiological studies, relatedness of isolated pathogenic bacterial strains can be determined from the number andsize of plasmids the bacteria carry. Plasmid profile analysis was among the earliest nucleic acid-based techniques applied to the diagnosis of infectious diseases and has proven useful in numerous investigations . This method has also been widely utilized for tracking antimicrobial resistance during nosocomial outbreaks. In studies of the epidemiology of plasmids, analysis of restriction fragments has proved valuable. This technique is widely used to monitor the spread of resistance-encoding plasmids between organisms and between hospitals, communities, or even countries.The weakness of the analysis is inherent in the fact that plasmids are mobile, extrachromosomal elements, not part of the chromosomal genotype. Because plasmids can be spontaneously lost from or readily acquired by a host stain, epidemiologically related isolatescan exhibit different plasmid profiles(van et al., 2007).
Wednesday, November 16, 2011
PB19 infection in transplantation
The first report of PVB19 infection after transplantation was published in 1986 [7]. Since then, numerous cases of PVB19 infections after solid-organ transplan-tation (SOT) and hematopoietic stem cell transplantation (HSCT) have been reported. Anemia is the predominant clinical manifestation. However, PVB19 has also been associated with hepatitis, pneumonitis, myocarditis, and allograft dysfunction. Nonetheless, the full spectrum of clinical manifestations of PVB19 infection among transplantat recipients is not well characterized.
The suppression of the RBC population that clinically results in anemia as the hallmark of PVB19 infection is consistent with the cellular tropism of this virus [71]. PVB19 infects erythroid progenitor cells by binding to the receptor known as the P antigen [71]. Subsequent PVB19 replication in erythroid progenitor cells leads to cellular lysis [72], which is characteristically manifested as pure red cell aplasia on bone marrow examination.
Immunocompetent individuals respond to PVB19 infection by producing virus-specific Ig [73–75]. Experimental studies have demonstrated that the generation of PVB19- specific Ig is temporally accompanied by reduction in the degree of parvoviremia [75]. The impairment in immunity that results
from pharmacologic immunosuppression limits the ability of transplant patients to produce neutralizing antibody, which leads to persistent PVB19 infection that manifests as chronicanemia. Not surprisingly, almost all patients in transplants patients series had chronic anemia, many patients did not possess PVB19-specific Ig at the onset of clinical disease, and almost all transplant patients without PVB19 IgM had parvoviremia. Demonstrates that the spectrum of clinical illness related to PVB19 is broad. This reflects the ability of PVB19 to infect other cells [76]. The cardiotropism of PVB19 is suggested by its association with myocarditis [77–79] and left ventricular
dysfunction [80] and by the demonstration of PVB19 DNA in fetal myocardial cells [81]. These data support the suggestion that myocarditis may occur in transplant patients with PVB19 disease, and this may be misdiagnosed as acute rejection and could result in death from cardiogenic shock [17, 28, 43]. The most likely cardiac target of PVB19 is the endothelium [81–83], because endothelial cells in small cardiac vessels also carry P antigen [54]. Likewise, endothelial infection could serve as the mechanism for PVB19-associated thrombotic microangiopathy [54].
Studies of parvoviruses that infect animals demonstrate the virions in various organs [84]. Parvovirus related to Aleutian mink disease was detected in alveolar cells in mink with acute interstitial pneumonitis [85]. Intact Aleutian mink disease parvoviral DNA has also been detected in glomeruli [85]. These animal data support the suggestion that PVB19 is a potential cause of pneumonitis [22, 31, 68], hepatitis [28, 58, 63, 66, 86], and collapsing glomerulopathy [26] in humans. Indeed, PVB19 has been demonstrated in the renal tissue and blood of patients with collapsing glomerulopathy and in hepatocytes of a patient with fibrosing cholestatic hepatitis [58]. Nevertheless, the reported associations between PVB19 and organ-specific syndromes do not definitely indicate causality.
The inability of transplant patients to mount sufficient anti-PVB19 Ig could present a diagnostic dilemma and delay treatment in patients seen at centers who rely on serological examination for the diagnosis PVB19. All except 1 of the patients who did not have PVB19 IgM detected had positive PCR assay results, suggesting the clinical utility of this molecular assay. Our
observation suggests that a negative PVB19 IgM serological test result does not rule out the diagnosis of PVB19 infection, and PCR should be used whenever a diagnosis of acute PVB19 infection is suspected in immunocompromised patients. Among patients who are highly suspected to have PVB19 disease but whose peripheral blood PCR assay result is negative, the diagnosis may be confirmed by bone marrow examination.
If feasible, reduction in immunosuppression should be a part of the treatment of PVB19 disease. Theoretically, this would allow the immune system to mount specific immunity against PBV19. The observation that parvoviremia ceases with generation of Ig [75] led to the current practice of intravenous Ig
treatment of PVB19. Intravenous Ig contains PVB19-specific antibodies. However, the dose and duration of treatment are not standardized. Clinical relapses are commonly observed (i.e. 1 relapse occurs for every 4–5 patients treated), which suggests that the patient experiences a continued state of severe immunosuppression and that there is a need to further reduceimmunosuppression or administer intravenous Ig for a longer period to neutralize parvoviremia. The rarity of this infection limits the conduct of a prospective trial to assess the optimal dose and duration of treatment.
In conclusion, PVB19 can cause rare but significant infectious complication after transplantation. The predominant clinical manifestation of PVB19 disease is anemia, although organ invasive manifestations, such as hepatitis, myocarditis, and pneumonitis, can be observed. However, whether these organ specific syndromes are causally linked to PVB19 infection remains to be proven. A high index of suspicion is advised when patients present with refractory and severe anemia after transplantation. In this clinical setting, PVB19 infection should be considered in the differential diagnosis, together with the other, more likely causes, such as an adverse reaction to treatment,blood loss, and anti-erythropoietin antibody, among others. In this regard, PCR may be a more useful noninvasive test for the confirmation of the diagnosis, because the PVB19 serological test results of many transplant patients are negative at the onset of clinical disease.
A retrospective study of parvovirus B19 antibody titres 2 to 3 years after bone marrow transplantation showed persisting IgG, suggesting that persistence of B19 antibody depends on prior recipient, but not donor, immunity.1
allogeneic peripheral blood stem cell transplantation (PBSCT(consistently results in severe immunodeficiency. It is known that human parvovirus B19 can persist in red blood cell precursors in the bone marrow of immunocompromised patients. An infection can cause severe complications including chronic bone marrow failure or pure red cell aplasia because of the inability of patients to produce neutralizing antibodies against the virus.3 The major route of transmission of parvovirus B19 is inhalation of respiratory droplets from infected people. Both of these patients were treated in special air-filter rooms designed to create a pathogen-reduced environment and no clinical infections were in family members or hospital staff. There is evidence for parvovirus B19 transmission via blood products, PBSC or bone marrow.4, 5 Patients required erythrocyte infusions during conditioning before transplantation. Parvovirus B19 is known to be a frequent contaminant of blood products,6 but as parvovirus B19 screening is not part of the routine control of blood products is not routinely followed in many countriesand so infection via this route cannot be excluded. Another route of infections in those patients is reactivation of old infection. with parvovirus B19 during immunosuppressive conditioning and further immunosuppression. This concept is supported by several reports both in PBSCT and in solid organ transplantation.7
parvovirus B19-induces erythematous infection in immuncompromised patients early after PBSC transplantation and should be considered in the differential diagnosis of acute GvHD of the skin. It highlights the importance of excluding the possibility of a viral infection before initiating treatment for acute GvHD. Furthermore, parvovirus B19 infection should be considered in cases of late anaemia after PBSC or bone marrow transplantation, occurring in patients known to be seropositive for parvovirus B19 IgG.
Lectures on applied clinical microbiology
http://www.amazon.com/dp/B004Y0XF54
The suppression of the RBC population that clinically results in anemia as the hallmark of PVB19 infection is consistent with the cellular tropism of this virus [71]. PVB19 infects erythroid progenitor cells by binding to the receptor known as the P antigen [71]. Subsequent PVB19 replication in erythroid progenitor cells leads to cellular lysis [72], which is characteristically manifested as pure red cell aplasia on bone marrow examination.
Immunocompetent individuals respond to PVB19 infection by producing virus-specific Ig [73–75]. Experimental studies have demonstrated that the generation of PVB19- specific Ig is temporally accompanied by reduction in the degree of parvoviremia [75]. The impairment in immunity that results
from pharmacologic immunosuppression limits the ability of transplant patients to produce neutralizing antibody, which leads to persistent PVB19 infection that manifests as chronicanemia. Not surprisingly, almost all patients in transplants patients series had chronic anemia, many patients did not possess PVB19-specific Ig at the onset of clinical disease, and almost all transplant patients without PVB19 IgM had parvoviremia. Demonstrates that the spectrum of clinical illness related to PVB19 is broad. This reflects the ability of PVB19 to infect other cells [76]. The cardiotropism of PVB19 is suggested by its association with myocarditis [77–79] and left ventricular
dysfunction [80] and by the demonstration of PVB19 DNA in fetal myocardial cells [81]. These data support the suggestion that myocarditis may occur in transplant patients with PVB19 disease, and this may be misdiagnosed as acute rejection and could result in death from cardiogenic shock [17, 28, 43]. The most likely cardiac target of PVB19 is the endothelium [81–83], because endothelial cells in small cardiac vessels also carry P antigen [54]. Likewise, endothelial infection could serve as the mechanism for PVB19-associated thrombotic microangiopathy [54].
Studies of parvoviruses that infect animals demonstrate the virions in various organs [84]. Parvovirus related to Aleutian mink disease was detected in alveolar cells in mink with acute interstitial pneumonitis [85]. Intact Aleutian mink disease parvoviral DNA has also been detected in glomeruli [85]. These animal data support the suggestion that PVB19 is a potential cause of pneumonitis [22, 31, 68], hepatitis [28, 58, 63, 66, 86], and collapsing glomerulopathy [26] in humans. Indeed, PVB19 has been demonstrated in the renal tissue and blood of patients with collapsing glomerulopathy and in hepatocytes of a patient with fibrosing cholestatic hepatitis [58]. Nevertheless, the reported associations between PVB19 and organ-specific syndromes do not definitely indicate causality.
The inability of transplant patients to mount sufficient anti-PVB19 Ig could present a diagnostic dilemma and delay treatment in patients seen at centers who rely on serological examination for the diagnosis PVB19. All except 1 of the patients who did not have PVB19 IgM detected had positive PCR assay results, suggesting the clinical utility of this molecular assay. Our
observation suggests that a negative PVB19 IgM serological test result does not rule out the diagnosis of PVB19 infection, and PCR should be used whenever a diagnosis of acute PVB19 infection is suspected in immunocompromised patients. Among patients who are highly suspected to have PVB19 disease but whose peripheral blood PCR assay result is negative, the diagnosis may be confirmed by bone marrow examination.
If feasible, reduction in immunosuppression should be a part of the treatment of PVB19 disease. Theoretically, this would allow the immune system to mount specific immunity against PBV19. The observation that parvoviremia ceases with generation of Ig [75] led to the current practice of intravenous Ig
treatment of PVB19. Intravenous Ig contains PVB19-specific antibodies. However, the dose and duration of treatment are not standardized. Clinical relapses are commonly observed (i.e. 1 relapse occurs for every 4–5 patients treated), which suggests that the patient experiences a continued state of severe immunosuppression and that there is a need to further reduceimmunosuppression or administer intravenous Ig for a longer period to neutralize parvoviremia. The rarity of this infection limits the conduct of a prospective trial to assess the optimal dose and duration of treatment.
In conclusion, PVB19 can cause rare but significant infectious complication after transplantation. The predominant clinical manifestation of PVB19 disease is anemia, although organ invasive manifestations, such as hepatitis, myocarditis, and pneumonitis, can be observed. However, whether these organ specific syndromes are causally linked to PVB19 infection remains to be proven. A high index of suspicion is advised when patients present with refractory and severe anemia after transplantation. In this clinical setting, PVB19 infection should be considered in the differential diagnosis, together with the other, more likely causes, such as an adverse reaction to treatment,blood loss, and anti-erythropoietin antibody, among others. In this regard, PCR may be a more useful noninvasive test for the confirmation of the diagnosis, because the PVB19 serological test results of many transplant patients are negative at the onset of clinical disease.
A retrospective study of parvovirus B19 antibody titres 2 to 3 years after bone marrow transplantation showed persisting IgG, suggesting that persistence of B19 antibody depends on prior recipient, but not donor, immunity.1
allogeneic peripheral blood stem cell transplantation (PBSCT(consistently results in severe immunodeficiency. It is known that human parvovirus B19 can persist in red blood cell precursors in the bone marrow of immunocompromised patients. An infection can cause severe complications including chronic bone marrow failure or pure red cell aplasia because of the inability of patients to produce neutralizing antibodies against the virus.3 The major route of transmission of parvovirus B19 is inhalation of respiratory droplets from infected people. Both of these patients were treated in special air-filter rooms designed to create a pathogen-reduced environment and no clinical infections were in family members or hospital staff. There is evidence for parvovirus B19 transmission via blood products, PBSC or bone marrow.4, 5 Patients required erythrocyte infusions during conditioning before transplantation. Parvovirus B19 is known to be a frequent contaminant of blood products,6 but as parvovirus B19 screening is not part of the routine control of blood products is not routinely followed in many countriesand so infection via this route cannot be excluded. Another route of infections in those patients is reactivation of old infection. with parvovirus B19 during immunosuppressive conditioning and further immunosuppression. This concept is supported by several reports both in PBSCT and in solid organ transplantation.7
parvovirus B19-induces erythematous infection in immuncompromised patients early after PBSC transplantation and should be considered in the differential diagnosis of acute GvHD of the skin. It highlights the importance of excluding the possibility of a viral infection before initiating treatment for acute GvHD. Furthermore, parvovirus B19 infection should be considered in cases of late anaemia after PBSC or bone marrow transplantation, occurring in patients known to be seropositive for parvovirus B19 IgG.
Lectures on applied clinical microbiology
http://www.amazon.com/dp/B004Y0XF54
Tuesday, November 15, 2011
Pseudomonas aeruginosa resistant to carbapenems
Ps. aeruginosa is an opportunistic pathogen that can cause severe invasive disease in critically ill and immunocompromised patients. This microorganism is an important cause of nosocomial infections, including pneumonia, wound infections, bacteremia and urinary tract infection (Ceza´ rio et al., 2009). The organism has both an intrinsic and acquired resistance to many antimicrobials and treatment of infection by this organism is difficult. Carbapenems have a high potency against a wide range of organisms and are one of the most active groups of β-lactam antibiotics against Ps. aeruginosa (Sakyo et al., 2006). Carbapenems are commonly used as last resort drugs for treatment of infections caused by multiresistant Ps. aeruginosa isolates. Carbapenem resistance was driven mainly by mutation mediated resistance leading to the repression or inactivation of the porin OprD, conferring resistance to imipenem and reduced susceptibility to meropenem (Quale et al., 2006), and by mutation leading to the hyperexpression of the chromosomally encoded cephalosporinase AmpC. Also remarkable, mutations leading to the up-regulation of one of the several efflux pumps encoded in the Ps. aeruginosa genome may confer resistance to multiple agents, including all β-lactams (meropenem is a substrate of efflux pumps whereas imipenem is not due to the absence of heterocyclic side chain), fluoroquinolones, and aminoglycosides. MexA-MexB-OprM is regarded the most efficient efflux pump for extrusion of carbapenems. Furthermore, the accumulation of various combinations of these chromosomal mutations can certainly lead to the emergence of multidrug resistant (Cavallo et al., 2007).
In addition to the mutation-mediated resistance, the presence of horizontally acquired resistance determinants in Ps. aeruginosa has been increasingly reported over the last decade. Among the certainly noteworthy determinants are those encoding MBL, particularly IMP and VIM enzymes, which are able to hydrolyze efficiently all β-lactams with the exception of aztreonam (Gutierrez et al., 2007). Although majority of calss A serine carbapenemases are found in Enterobacteriaceae family members, but resistance may transfer through mobile genetic elements to other families such as GES enzyme which may be found as cassettes within integrons on plasmid mostly in Ps. aeruginosa (Overturf, 2010).
In addition to the mutation-mediated resistance, the presence of horizontally acquired resistance determinants in Ps. aeruginosa has been increasingly reported over the last decade. Among the certainly noteworthy determinants are those encoding MBL, particularly IMP and VIM enzymes, which are able to hydrolyze efficiently all β-lactams with the exception of aztreonam (Gutierrez et al., 2007). Although majority of calss A serine carbapenemases are found in Enterobacteriaceae family members, but resistance may transfer through mobile genetic elements to other families such as GES enzyme which may be found as cassettes within integrons on plasmid mostly in Ps. aeruginosa (Overturf, 2010).
Monday, November 14, 2011
Nanotchnology and Treatment of Bacterial Infections
The new age drugs are nanoparticles of polymers, metals or ceramics, which can combat conditions like cancer [83] and fight human pathogens like bacteria [84-88].
The development of new resistant strains of bacteria to current antibiotics [89] has become a serious problem in public health; therefore, there is a strong incentive to develop new bactericides [86]. Bacteria have different membrane structures which allow a general classification of them as Gram-negative or Gram positive. The structural differences lie in the organization of a key component of the membrane, peptidoglycan. Gram negative bacteria exhibit only a thin peptidoglycan layer (~2–3 nm) between the cytoplasmic membrane and the outer membrane [90]; in contrast, Gram-positive bacteria lack the outer membrane but have a peptidoglycan layer of about 30 nm thick [91].
Silver has long been known to exhibit a strong toxicity to a wide range of micro-organisms [92]; for this reason silver-based compounds have been used extensively in many bactericidal applications [93, 94]. Silver compounds have also been used in the medical field to treat burns and a variety of infections [95]. Several salts of silver and their derivatives are commercially employed as antimicrobial agents [96]. Commendable efforts have been made to explore this property using electron microscopy, which has revealed size dependent interaction of silver nanoparticles with bacteria [87]. Nanoparticles of silver have thus been studied as a medium for antibiotic delivery [97], and to synthesize composites for use as disinfecting filters [98] and coating materials [99]. However, the bactericidal property of these nanoparticles depends on their stability in the growth medium, since this imparts greater retention time for bacterium–nanoparticle interaction. There lies a strong challenge in preparing nanoparticles of silver stable enough to significantly restrict bacterial growth.
Studies were carried out on both antibiotic resistant (ampicillin- resistant) and nonresistant strains of gram-negative (Escherichia coli) and a non-resistant strain of gram-positive bacteria (Staphylococcus aureus). A multi-drug resistant strain of gram-negative (Salmonella typhus, resistant to chloramphenicol, amoxycilin and trimethoprim) bacteria was also subjected to analysis to examine the antibacterial effect of the nanoparticles [100]. Efforts have been made to understand the underlying molecular mechanism of such antimicrobial actions. The effect of the nanoparticles was found to be significantly more pronounced on the gram-negative strains, irrespective of whether the strains were resistant or not, than on the gram-positive organisms. This could be attributed this enhanced antibacterial effect of the nanoparticles to their stability in the medium as a colloid, which modulates the phosphotyrosine profile of the bacterial proteins and arrests bacterial growth.
The bactericidal effect of silver ions on micro-organisms is very well known; however, the bactericidal mechanism is only partially understood. It has been proposed that ionic silver strongly interacts with thiol groups of vital enzymes and inactivates them (101, 102). Experimental evidence suggests that DNA loses its replication ability once the bacteria have been treated with silver ions [95]. Other studies have shown evidence of structural changes in the cell membrane as well as the formation of small electron-dense granules formed by silver and sulfur [95, 103].
Silver ions have been demonstrated to be useful and effective in bactericidal applications, but due to the unique properties of nanoparticles nanotechnology presents a reasonable alternative for development of new bactericides. Metal particles in the nanometer size range exhibit physical properties that are different from both the ion and the bulk material. This makes them exhibit remarkable properties such as increased catalytic activity due to morphologies with highly active facets [104-109]. Several electron microscopy techniques can be applied to study the mechanism by which silver nanoparticles interact with these bacteria. We can use high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM), and developed a novel sample preparation that avoids the use of heavy metal based compounds such as OsO4. High resolutions and more accurate x-ray microanalysis were obtained.
Generally, the encapsulation of antibiotics in liposomes or in nanoparticles increased the maximal tolerated dose and the therapeutic index of the antibiotics compared with the free drug. This can be explained by a modification of the pharmacokinetic profile of the antibiotic when encapsulated as well as by a modification of its bio distribution. For instance, in a liposome formulation of amikacin (Mikasome\, Gilead), which is in clinic evaluation, the antibiotic was found 2- to 6-fold more active than the free drug and the free streptomycin in an acute experimental model of murine tuberculosis in which bacteria were located into macrophages.
In a model of mice infected by Mycobacterium avium, amikacin in liposomes could reduce viable bacterial count in liver, spleen, and, to a lesser extent, lungs by approximately 3-log10 compared with the untreated control.
In another example, the entrapment of ampicillin in poly(isobutylcyanoacrylate) nanoparticles increased by 120-fold the efficacy of the antibiotic in an experimental acute infection of mice by Salmonella typhi murium. In this model, 100% of the infected mice treated with a single dose of the nanoparticles survived, whereas all the untreated animals died after 10 days. Such high activity was explained by a complete sterilization of the organs where the intracellular bacteria were located. Treatment with liposomes was less efficient. The survival of mice did not exceed 60%, and the infected organs were never completely sterilized in mice that survived.
Ampicillin-loaded nanoparticles were also found more efficient than liposomes for the treatment of listeriosis in a model of chronic infection of mice by Listeria monocytogenes [188]. In this case, it was shown that the spleen was not totally sterilized, and a reinfection occurred after several days whatever the treatment was. In this model, reinfection was believed to occur from non dividing bacteria, but even nanoparticles loaded with ciprofloxacine, a fluoroquinolone with antibacterial activity against both dividing and nondividing bacteria, could not totally eradicate the infectious reservoir [110].
This shows the extreme difficulty to eradicate all bacteria from the body even when they are a priori located in the MPS. Another difficulty is to reach infections, which develop outside the MPS and outside macrophages. Indeed, it was suggested that part of the clinical trials, which aimed to treat patients infected by tuberculosis with Mikasome, failed because the antibiotic was released in macrophages that were too far from the extracellular bacilli clustered in cavity caseum in the human infection [111]. In case of tuberculosis, it is now known that targeting the non replicated persistent bacilli still remains a challenge to be addressed [112]. Further improvements of drug delivery systems are still needed to enhance the targeting of the extracellular infectious sites.
So far, most of the very promising data were obtained by treating experimental animal infections with antibiotics associated with nanodevices in comparison with the free drug. However, in front of the somewhat disappointing results obtained with Mikasome\ during clinical trials, questions about the relevance of the animal experimental models (with intact host defense and with highly susceptible bacteria to the antibiotics) were raised.
Indeed, in clinical practice, treatments are often given to patients with impaired host defenses and who may be infected with bacteria of low antibiotic susceptibility. Only a few studies considered experimental models on animals with impaired host defenses [110].
In vitro models must also be handled cautiously because they were not always predictive of the in vivo activity. Indeed, the activity measured in vitro may be found dramatically reduced or significantly promoted because of synergies with lymphocytes when tested in vivo in animal models [113]. Nevertheless, for a systemic treatment of bacterial infection in which the target cells are the MPS macrophages of the liver and the spleen, conventional liposomes and nanoparticles can be suggested as the most relevant delivery systems for antibiotics. The efficacy of liposomes was found very dependent on their physicochemical characteristics. For instance, specific composition may affect the bactericidal activity by interaction with the infected organism [114].
In contrast, such formulations seemed of limited value to treat infections in which bacteria are located outside the main MPS organs (i.e., liver, spleen, and bone marrow), and more efforts are still required to address this goal [115].
Indeed, targeted systems to extracellular bacteria and to other reservoir organs may contribute to make progress in the battle against bacterial infections. It is also needed to develop appropriate strategies to eliminate persistent bacteria, which are either in inaccessible sites or in a state of dormancy within macrophages. Some attempts were made using targeted liposomes with mannose to promote recognition by human phagocytic cells [116]. However, the design of a targeted device seemed very delicate to find the right length of spacer between the targeting moiety and the surface of the device and to balance between the numbers of mannose residues on the lipid surface.
Finally, only a few investigations have considered comparative experiments performed with liposomes and other nanosystems. The nanoparticles seemed more efficient than niosomes, which were, in turn, more efficient than liposomes [117,118]. This superiority of nanoparticles may be explained by a higher stability in biological media. In the future, the problem of stability of delivery systems in biological fluid may become even more important in view of the systemic delivery of targeted antibiotics by the oral route. This is another challenge that emerged and is still poorly documented at the moment [119].
Liposome formulations of antibiotics were also evaluated for the local delivery of antibiotics to be used as controlled release system at the site of the infection. They have proven to be of interest for readily accessible infected tissues such as the eye, wound, and lungs [120]. This strategy was suggested in surgical wound prophylaxis [121,122], in the treatment of keratitis using liposomal formulation of tobramycin in eye drops (123), in the treatment of endophthalmitis by intravitreal injection of amikacin-loaded liposomes, and in lung infections by aerosol delivery of the liposomal formulation of antibiotics [124,125]. Recently, a bioresorbable composite pellet of calcium sulfate and hydroxyapatite nanoparticles was studied as a material for local and sustained delivery of antibiotics in bone infections [126]. In this system, the nanoparticles of hydroxyapatite changed the properties of the material by increasing the specific surface of the device and by allowing a higher loading of antibiotics. The nanoparticles incorporated in the material could also advantageously modify the released profile of antibiotics permitting the release of the total dose of the antibiotic incorporated in the material at the end of the process. This was actually not the case with the material devoid of nanoparticles, which retained up to 25% of the dose of the antibiotic after 10 days.
Finally, the tolerance of the material modified by the nanoparticles was improved because the quantity of acid produced by the dissolution of calcium sulfate and responsible for an inflammatory response was reduced. This example illustrates advantages brought when nanotechnology is associated with other technologies to improve the pharmacological properties of a material used as an implant.
Beside what could be considered as Bartificial^ nanotechnologies including liposomes and nanoparticles, some authors considered the use of Bnatural^ nanotechnologies to fight against resistant bacteria by using bacteriophages. This approach was used once in human with an unexpected success in combination with ciprofloxacin for local treatment of patients with wounds infected by multidrug-resistant Staphylococcus aureus [127].
Data obtained on infected animal models suggested that bacterial infection can be circumvented only with functional phage specific to the bacterial strain [128]. The formidable activity observed was suggested to result from the functional capability of the phage only and not due to a nonspecific immune effect of the host defense [129]. At the moment, no side effect was reported about the use of phages, but the number of studies remained very limited. The level of antibodies against phages found in the rescued animals was not substantially elevated.
The development of new resistant strains of bacteria to current antibiotics [89] has become a serious problem in public health; therefore, there is a strong incentive to develop new bactericides [86]. Bacteria have different membrane structures which allow a general classification of them as Gram-negative or Gram positive. The structural differences lie in the organization of a key component of the membrane, peptidoglycan. Gram negative bacteria exhibit only a thin peptidoglycan layer (~2–3 nm) between the cytoplasmic membrane and the outer membrane [90]; in contrast, Gram-positive bacteria lack the outer membrane but have a peptidoglycan layer of about 30 nm thick [91].
Silver has long been known to exhibit a strong toxicity to a wide range of micro-organisms [92]; for this reason silver-based compounds have been used extensively in many bactericidal applications [93, 94]. Silver compounds have also been used in the medical field to treat burns and a variety of infections [95]. Several salts of silver and their derivatives are commercially employed as antimicrobial agents [96]. Commendable efforts have been made to explore this property using electron microscopy, which has revealed size dependent interaction of silver nanoparticles with bacteria [87]. Nanoparticles of silver have thus been studied as a medium for antibiotic delivery [97], and to synthesize composites for use as disinfecting filters [98] and coating materials [99]. However, the bactericidal property of these nanoparticles depends on their stability in the growth medium, since this imparts greater retention time for bacterium–nanoparticle interaction. There lies a strong challenge in preparing nanoparticles of silver stable enough to significantly restrict bacterial growth.
Studies were carried out on both antibiotic resistant (ampicillin- resistant) and nonresistant strains of gram-negative (Escherichia coli) and a non-resistant strain of gram-positive bacteria (Staphylococcus aureus). A multi-drug resistant strain of gram-negative (Salmonella typhus, resistant to chloramphenicol, amoxycilin and trimethoprim) bacteria was also subjected to analysis to examine the antibacterial effect of the nanoparticles [100]. Efforts have been made to understand the underlying molecular mechanism of such antimicrobial actions. The effect of the nanoparticles was found to be significantly more pronounced on the gram-negative strains, irrespective of whether the strains were resistant or not, than on the gram-positive organisms. This could be attributed this enhanced antibacterial effect of the nanoparticles to their stability in the medium as a colloid, which modulates the phosphotyrosine profile of the bacterial proteins and arrests bacterial growth.
The bactericidal effect of silver ions on micro-organisms is very well known; however, the bactericidal mechanism is only partially understood. It has been proposed that ionic silver strongly interacts with thiol groups of vital enzymes and inactivates them (101, 102). Experimental evidence suggests that DNA loses its replication ability once the bacteria have been treated with silver ions [95]. Other studies have shown evidence of structural changes in the cell membrane as well as the formation of small electron-dense granules formed by silver and sulfur [95, 103].
Silver ions have been demonstrated to be useful and effective in bactericidal applications, but due to the unique properties of nanoparticles nanotechnology presents a reasonable alternative for development of new bactericides. Metal particles in the nanometer size range exhibit physical properties that are different from both the ion and the bulk material. This makes them exhibit remarkable properties such as increased catalytic activity due to morphologies with highly active facets [104-109]. Several electron microscopy techniques can be applied to study the mechanism by which silver nanoparticles interact with these bacteria. We can use high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM), and developed a novel sample preparation that avoids the use of heavy metal based compounds such as OsO4. High resolutions and more accurate x-ray microanalysis were obtained.
Generally, the encapsulation of antibiotics in liposomes or in nanoparticles increased the maximal tolerated dose and the therapeutic index of the antibiotics compared with the free drug. This can be explained by a modification of the pharmacokinetic profile of the antibiotic when encapsulated as well as by a modification of its bio distribution. For instance, in a liposome formulation of amikacin (Mikasome\, Gilead), which is in clinic evaluation, the antibiotic was found 2- to 6-fold more active than the free drug and the free streptomycin in an acute experimental model of murine tuberculosis in which bacteria were located into macrophages.
In a model of mice infected by Mycobacterium avium, amikacin in liposomes could reduce viable bacterial count in liver, spleen, and, to a lesser extent, lungs by approximately 3-log10 compared with the untreated control.
In another example, the entrapment of ampicillin in poly(isobutylcyanoacrylate) nanoparticles increased by 120-fold the efficacy of the antibiotic in an experimental acute infection of mice by Salmonella typhi murium. In this model, 100% of the infected mice treated with a single dose of the nanoparticles survived, whereas all the untreated animals died after 10 days. Such high activity was explained by a complete sterilization of the organs where the intracellular bacteria were located. Treatment with liposomes was less efficient. The survival of mice did not exceed 60%, and the infected organs were never completely sterilized in mice that survived.
Ampicillin-loaded nanoparticles were also found more efficient than liposomes for the treatment of listeriosis in a model of chronic infection of mice by Listeria monocytogenes [188]. In this case, it was shown that the spleen was not totally sterilized, and a reinfection occurred after several days whatever the treatment was. In this model, reinfection was believed to occur from non dividing bacteria, but even nanoparticles loaded with ciprofloxacine, a fluoroquinolone with antibacterial activity against both dividing and nondividing bacteria, could not totally eradicate the infectious reservoir [110].
This shows the extreme difficulty to eradicate all bacteria from the body even when they are a priori located in the MPS. Another difficulty is to reach infections, which develop outside the MPS and outside macrophages. Indeed, it was suggested that part of the clinical trials, which aimed to treat patients infected by tuberculosis with Mikasome, failed because the antibiotic was released in macrophages that were too far from the extracellular bacilli clustered in cavity caseum in the human infection [111]. In case of tuberculosis, it is now known that targeting the non replicated persistent bacilli still remains a challenge to be addressed [112]. Further improvements of drug delivery systems are still needed to enhance the targeting of the extracellular infectious sites.
So far, most of the very promising data were obtained by treating experimental animal infections with antibiotics associated with nanodevices in comparison with the free drug. However, in front of the somewhat disappointing results obtained with Mikasome\ during clinical trials, questions about the relevance of the animal experimental models (with intact host defense and with highly susceptible bacteria to the antibiotics) were raised.
Indeed, in clinical practice, treatments are often given to patients with impaired host defenses and who may be infected with bacteria of low antibiotic susceptibility. Only a few studies considered experimental models on animals with impaired host defenses [110].
In vitro models must also be handled cautiously because they were not always predictive of the in vivo activity. Indeed, the activity measured in vitro may be found dramatically reduced or significantly promoted because of synergies with lymphocytes when tested in vivo in animal models [113]. Nevertheless, for a systemic treatment of bacterial infection in which the target cells are the MPS macrophages of the liver and the spleen, conventional liposomes and nanoparticles can be suggested as the most relevant delivery systems for antibiotics. The efficacy of liposomes was found very dependent on their physicochemical characteristics. For instance, specific composition may affect the bactericidal activity by interaction with the infected organism [114].
In contrast, such formulations seemed of limited value to treat infections in which bacteria are located outside the main MPS organs (i.e., liver, spleen, and bone marrow), and more efforts are still required to address this goal [115].
Indeed, targeted systems to extracellular bacteria and to other reservoir organs may contribute to make progress in the battle against bacterial infections. It is also needed to develop appropriate strategies to eliminate persistent bacteria, which are either in inaccessible sites or in a state of dormancy within macrophages. Some attempts were made using targeted liposomes with mannose to promote recognition by human phagocytic cells [116]. However, the design of a targeted device seemed very delicate to find the right length of spacer between the targeting moiety and the surface of the device and to balance between the numbers of mannose residues on the lipid surface.
Finally, only a few investigations have considered comparative experiments performed with liposomes and other nanosystems. The nanoparticles seemed more efficient than niosomes, which were, in turn, more efficient than liposomes [117,118]. This superiority of nanoparticles may be explained by a higher stability in biological media. In the future, the problem of stability of delivery systems in biological fluid may become even more important in view of the systemic delivery of targeted antibiotics by the oral route. This is another challenge that emerged and is still poorly documented at the moment [119].
Liposome formulations of antibiotics were also evaluated for the local delivery of antibiotics to be used as controlled release system at the site of the infection. They have proven to be of interest for readily accessible infected tissues such as the eye, wound, and lungs [120]. This strategy was suggested in surgical wound prophylaxis [121,122], in the treatment of keratitis using liposomal formulation of tobramycin in eye drops (123), in the treatment of endophthalmitis by intravitreal injection of amikacin-loaded liposomes, and in lung infections by aerosol delivery of the liposomal formulation of antibiotics [124,125]. Recently, a bioresorbable composite pellet of calcium sulfate and hydroxyapatite nanoparticles was studied as a material for local and sustained delivery of antibiotics in bone infections [126]. In this system, the nanoparticles of hydroxyapatite changed the properties of the material by increasing the specific surface of the device and by allowing a higher loading of antibiotics. The nanoparticles incorporated in the material could also advantageously modify the released profile of antibiotics permitting the release of the total dose of the antibiotic incorporated in the material at the end of the process. This was actually not the case with the material devoid of nanoparticles, which retained up to 25% of the dose of the antibiotic after 10 days.
Finally, the tolerance of the material modified by the nanoparticles was improved because the quantity of acid produced by the dissolution of calcium sulfate and responsible for an inflammatory response was reduced. This example illustrates advantages brought when nanotechnology is associated with other technologies to improve the pharmacological properties of a material used as an implant.
Beside what could be considered as Bartificial^ nanotechnologies including liposomes and nanoparticles, some authors considered the use of Bnatural^ nanotechnologies to fight against resistant bacteria by using bacteriophages. This approach was used once in human with an unexpected success in combination with ciprofloxacin for local treatment of patients with wounds infected by multidrug-resistant Staphylococcus aureus [127].
Data obtained on infected animal models suggested that bacterial infection can be circumvented only with functional phage specific to the bacterial strain [128]. The formidable activity observed was suggested to result from the functional capability of the phage only and not due to a nonspecific immune effect of the host defense [129]. At the moment, no side effect was reported about the use of phages, but the number of studies remained very limited. The level of antibodies against phages found in the rescued animals was not substantially elevated.
Application of Nano Gold and Nano Silver in Clinical Microbiology
Recently, nanoparticle-based assay has been introduced as a tool for both disease therapy and laboratory diagnosis [49-52]. The detection of Salmonella spp. using antibody-coated gold nanoparticles and dielectrophoretic impedance measurement has been reported [53]. It has been also demonstrated that Leptospira antibody-coated gold nanoparticles can be used for urine Leptospira detection. Nano gold particles can be also used for drug vectorization and DNA/gene delivery for various diseases, in particular cancer, Alzheimer, HIV, hepatitis, tuberculosis, arthritis, and diabetes
Metallic nanoparticles (e.g. gold and silver colloids) have recently been successfully used as labels technology because of their easily controllable size, unique optical and electrical properties, and high biocompatibility with antibodies, proteins, RNA, and DNA [54,55]
Recently, Mirkin and co-workers developed a scanometric DNA array (56) an electrical detection-based DNA array[57], and Raman spectroscopic fingerprints for DNA and RNA detection [58] based on silver deposition on gold nanoparticles. Based on the same principle, Alexandre et al. (59) implemented the colorimetric method for DNA microarray detection with a colorimetric-based workstation containing a charge coupled device (CCD) camera. In addition, Chu et al. [60]used silver-enhanced ANP labels for an electrochemical stripping metalloimmunoassay.
Using electrical detection is a new technique for protein analysis. Kelev and Kalev [61] used the in situ assembly of colloidal particles onto micropatterned electrodes for biosensor as a specific proteins.. Recently, Yeh and his colleagues [62] constructed a model format is based on the sandwich immunoassay (three-layer format). The AgNPs were introduced into the electro-microchip by the specific binding of the antibodies and then coupled with silver enhancement solution to reduce silver ions to silver metal. The silver precipitation constructs a “bridge” between two electrodes of the electro-microchip allowing the electrons to pass. There was a significant difference in impedance between the experimental sample and the negative control after 10 min of reaction time. The proposed method requires less time and fewer steps than enzyme-linked immunosorbent assay (ELISA). It also has a high detection sensitivity (10 μg/mL of 1st antibody (IgG) immobilized on slides and 0.1 ng/mL of antigen (protein A)). Therefore, a new immunoassay is constructed using an electro-microchip, antibody–AgNPs conjugates, and a silver enhancement reaction.
Metallic nanoparticles (e.g. gold and silver colloids) have recently been successfully used as labels technology because of their easily controllable size, unique optical and electrical properties, and high biocompatibility with antibodies, proteins, RNA, and DNA [54,55]
Recently, Mirkin and co-workers developed a scanometric DNA array (56) an electrical detection-based DNA array[57], and Raman spectroscopic fingerprints for DNA and RNA detection [58] based on silver deposition on gold nanoparticles. Based on the same principle, Alexandre et al. (59) implemented the colorimetric method for DNA microarray detection with a colorimetric-based workstation containing a charge coupled device (CCD) camera. In addition, Chu et al. [60]used silver-enhanced ANP labels for an electrochemical stripping metalloimmunoassay.
Using electrical detection is a new technique for protein analysis. Kelev and Kalev [61] used the in situ assembly of colloidal particles onto micropatterned electrodes for biosensor as a specific proteins.. Recently, Yeh and his colleagues [62] constructed a model format is based on the sandwich immunoassay (three-layer format). The AgNPs were introduced into the electro-microchip by the specific binding of the antibodies and then coupled with silver enhancement solution to reduce silver ions to silver metal. The silver precipitation constructs a “bridge” between two electrodes of the electro-microchip allowing the electrons to pass. There was a significant difference in impedance between the experimental sample and the negative control after 10 min of reaction time. The proposed method requires less time and fewer steps than enzyme-linked immunosorbent assay (ELISA). It also has a high detection sensitivity (10 μg/mL of 1st antibody (IgG) immobilized on slides and 0.1 ng/mL of antigen (protein A)). Therefore, a new immunoassay is constructed using an electro-microchip, antibody–AgNPs conjugates, and a silver enhancement reaction.
Friday, November 11, 2011
Superbugs that commonly cause HAIs (2)
Clinically important superbugs that commonly cause healthcare-associated infections include methicillin resistant Staph aureus (MRSA), vancomycin-resistant enterococci (VRE), and multidrug-resistant Gram-negative rods, including strains of Pseudomonas aeruginosa and E. coli. Antimicrobial resistance among nosocomial pathogens often results in prolonged periods of antimicrobial therapy and increased treatment costs, prolonged hospital stays and higher mortality (Dohmen, 2008).
Gram-positive organisms:
Two health care-related pathogens continue to be of particular concern: MRSA and vancomycin-resistant enterococci (VRE). In some hospitals, over 70% of the Staph aureus isolated from inpatients are MRSA (Hu et al., 2009).
1. Staphylococcus aureus:
Since the introduction of penicillin in the late 1940s and throughout the antibiotic era to the present, S.aureus has adapted rapidly by becoming resistant to each new type of drug (Swartz, 1994).
By the late 1960s, more than 80% of both community- and hospital-acquired staphylococcal isolates were resistant to penicillin. This pattern of resistance, first emerging in hospitals and then spreading to the community, is now well established pattern that recurs with each new wave of antimicrobial resistance (Chambers, 2001).
Methicillin, the prototype penicillinase-resistant penicillin, became available in 1959 to treat staphylococci resistant to penicillin. Methicillin is one of several semisynthetic penicillins with bulky side chains, such that the antibiotic undergoes very slow hydrolysis by the staphylococcal beta-lactamase (Jevons et al., 1963).
Methicillin resistant strains of S. aureus were first detected in 1961 in a British hospital, shortly after the introduction of methicillin into clinical use in 1959. MRSA became a major problem in hospital settings worldwide in the 1980s (Boyce, 1994).
Need to read more
read Manual of Antibiotics: Method of Actions, Mechanisms of Resistance and Relations to Health Care associated Infections
http://www.amazon.com/Manual-Antibiotics-Mechanisms-Resistance-ebook/dp/B0050VQWXI
If you buy the first book, you can ask for the second part free (Pseudomonas spp., mechanisms of resistnce)
Gram-positive organisms:
Two health care-related pathogens continue to be of particular concern: MRSA and vancomycin-resistant enterococci (VRE). In some hospitals, over 70% of the Staph aureus isolated from inpatients are MRSA (Hu et al., 2009).
1. Staphylococcus aureus:
Since the introduction of penicillin in the late 1940s and throughout the antibiotic era to the present, S.aureus has adapted rapidly by becoming resistant to each new type of drug (Swartz, 1994).
By the late 1960s, more than 80% of both community- and hospital-acquired staphylococcal isolates were resistant to penicillin. This pattern of resistance, first emerging in hospitals and then spreading to the community, is now well established pattern that recurs with each new wave of antimicrobial resistance (Chambers, 2001).
Methicillin, the prototype penicillinase-resistant penicillin, became available in 1959 to treat staphylococci resistant to penicillin. Methicillin is one of several semisynthetic penicillins with bulky side chains, such that the antibiotic undergoes very slow hydrolysis by the staphylococcal beta-lactamase (Jevons et al., 1963).
Methicillin resistant strains of S. aureus were first detected in 1961 in a British hospital, shortly after the introduction of methicillin into clinical use in 1959. MRSA became a major problem in hospital settings worldwide in the 1980s (Boyce, 1994).
Need to read more
read Manual of Antibiotics: Method of Actions, Mechanisms of Resistance and Relations to Health Care associated Infections
http://www.amazon.com/Manual-Antibiotics-Mechanisms-Resistance-ebook/dp/B0050VQWXI
If you buy the first book, you can ask for the second part free (Pseudomonas spp., mechanisms of resistnce)
Causes of superbugs prevalence in HAIS
It is notable that the majority of infection control problems in the hospital are due to increasingly resistant bacteria. It is important to reflect on the role that antibiotic use may have as a selecting force for evolution of these superbugs. Selecting force of antibiotics, combined with lapses in infection control techniques make from these resistant organisms resident flora in hospitals and lead to their spread from person to person (Gould, 2009).
A-Selective pressure of antibiotic use:
Current policies to shorten length of stay and curtail costs encourage empiric use, often of unnecessarily broad-spectrum antibiotics. Combination therapy is often used for a number of reasons including broadening spectrum to accommodate increasing antibiotic resistance. This over-use and sometimes misuse inevitably leads to evolution and spread of superbugs (Gould, 2009).
Evolution of antibiotic resistance is the result of two essential forces: variability (chance) and selection (necessity). Variability is created by random mutation; variants with a mutation in the antibiotic target become resistant. These variants are selected by antibiotic use and consequently they increase the frequency of resistance. If the variability (as in a hyper-mutable strain) increased or the intensity of selection (antibiotic hyper-consumption) increased, the result is more resistance (Baquero and Cantón, 2009).
Indeed, antibiotic use not only selects for and maintains antibiotic resistance, but it also enhances its spread. This point is crucial to the control of modern HAIs and illustrates why traditional infection control policies have not been as successful as was hoped (Dancer, 2008).
Antimicrobials are the only category of drugs that have “societal” consequences. In other words, anti-hypertensives or lipid lowering agents only impact the person receiving these drugs. While in case of antimicrobials, in contrast, an individual can receive these drugs, develop resistance to them, and then pass along the newly created resistant organism to individuals that have never been exposed to these antimicrobials (Owens and Lautenbach, 2009).
Certain antibiotic classes are highly associated with colonization with superbugs compared to other antibiotic classes. The risk for colonization increases if broad-spectrum antibiotic is used or if the antibiotic is used in low doses over long periods. In the case of MRSA increased rates of MRSA colonization and infections are seen with glycopeptides, cephalosporins and quinolones therapies (Tacconelli, 2008).
B-Lapses in infection control techniques:
Reliance on antibiotics has become over-reliance, leading to poor quality infection control in the belief that infection has been beaten by antibiotics (Gould, 2007).
Cleaning is routinely monitored by visual assessment. Looking to see if a ward is clean does not provide a reliable assessment of the infection risk for an individual patient on that ward. The organisms that cause infection are invisible to the naked eye and their existence is not necessarily associated with the presence of visual dirt (Dancer, 2009).
The increase in superbugs appears to be linked to hospital cross-infection by these organisms. If staff enters a room containing MRSA patient, two-thirds of them will acquire the patient’s strain on gloved hands or apron (Dancer, 2009).
Poor hand hygiene by hospital staff has been associated with the spread of resistant organisms and an increase in hand washing results in decreased rates of these organisms (Girou et al., 2006).
You want to know more about antibiotics Read
Manual of Antibiotics: Method of Actions, Mechanisms of Resistance and Relations to Health Care associated Infections
http://www.amazon.com/Manual-Antibiotics-Mechanisms-Resistance-ebook/dp/B0050VQWXI
A-Selective pressure of antibiotic use:
Current policies to shorten length of stay and curtail costs encourage empiric use, often of unnecessarily broad-spectrum antibiotics. Combination therapy is often used for a number of reasons including broadening spectrum to accommodate increasing antibiotic resistance. This over-use and sometimes misuse inevitably leads to evolution and spread of superbugs (Gould, 2009).
Evolution of antibiotic resistance is the result of two essential forces: variability (chance) and selection (necessity). Variability is created by random mutation; variants with a mutation in the antibiotic target become resistant. These variants are selected by antibiotic use and consequently they increase the frequency of resistance. If the variability (as in a hyper-mutable strain) increased or the intensity of selection (antibiotic hyper-consumption) increased, the result is more resistance (Baquero and Cantón, 2009).
Indeed, antibiotic use not only selects for and maintains antibiotic resistance, but it also enhances its spread. This point is crucial to the control of modern HAIs and illustrates why traditional infection control policies have not been as successful as was hoped (Dancer, 2008).
Antimicrobials are the only category of drugs that have “societal” consequences. In other words, anti-hypertensives or lipid lowering agents only impact the person receiving these drugs. While in case of antimicrobials, in contrast, an individual can receive these drugs, develop resistance to them, and then pass along the newly created resistant organism to individuals that have never been exposed to these antimicrobials (Owens and Lautenbach, 2009).
Certain antibiotic classes are highly associated with colonization with superbugs compared to other antibiotic classes. The risk for colonization increases if broad-spectrum antibiotic is used or if the antibiotic is used in low doses over long periods. In the case of MRSA increased rates of MRSA colonization and infections are seen with glycopeptides, cephalosporins and quinolones therapies (Tacconelli, 2008).
B-Lapses in infection control techniques:
Reliance on antibiotics has become over-reliance, leading to poor quality infection control in the belief that infection has been beaten by antibiotics (Gould, 2007).
Cleaning is routinely monitored by visual assessment. Looking to see if a ward is clean does not provide a reliable assessment of the infection risk for an individual patient on that ward. The organisms that cause infection are invisible to the naked eye and their existence is not necessarily associated with the presence of visual dirt (Dancer, 2009).
The increase in superbugs appears to be linked to hospital cross-infection by these organisms. If staff enters a room containing MRSA patient, two-thirds of them will acquire the patient’s strain on gloved hands or apron (Dancer, 2009).
Poor hand hygiene by hospital staff has been associated with the spread of resistant organisms and an increase in hand washing results in decreased rates of these organisms (Girou et al., 2006).
You want to know more about antibiotics Read
Manual of Antibiotics: Method of Actions, Mechanisms of Resistance and Relations to Health Care associated Infections
http://www.amazon.com/Manual-Antibiotics-Mechanisms-Resistance-ebook/dp/B0050VQWXI
Prevention of hospital acquired influenza
Facilities should use a hierarchy of controls approach to prevent influenza transmission within healthcare settings. The hierarchy of controls to protect workers from occupational injury or illness places preventive interventions in groups that are ranked according to their likely effectiveness in reducing or removing the source of exposure. To apply the hierarchy of controls to prevent influenza transmission, facilities should follow the following steps, as shown in table (10) (Narain et al., 2009).
Thursday, November 10, 2011
Biological hazards and Laboratory work with Mycobacterium tuberculosis
It must also be emphasized that M. tuberculosis can survive for several days on inanimate surfaces. Survival of M. tuberculosis outside the host can be particularly long with, for example: 90 to 120 days on dust, 45 days on manure, 105 days on paper, 6 to 8 months in sputum (cool, dark location) and 45 days on clothing. Hence a work surface, that has not been properly disinfected, represents an additional source of moderate risk of transmission (Rubin, 1991).
Among the laboratory techniques used for the identification and characterization of mycobacteria, the following practices are likely to increase the risk of contamination or to generate infectious aerosols producing droplet nuclei (Jensen et al., 2005):
1) Handling of containers with clinical specimens: even if this situation is unlikely to generate aerosols, it is the initial step where laboratory personnel is potentially exposed to the tubercle bacilli. It was shown that the outside of containers used for collecting clinical specimens is frequently contaminated by M. tuberculosis (6.5%) or by other airborne pathogens (15%).
2) Centrifugations: fluid may spill from centrifuge tubes or tubes may break, releasing a large amount of aerosols.
3) Pipetting: pipettes and Pasteur pipettes in particular are likely to generate bubbles which burst and form aerosols.
4) Mechanical homogenizing (vortexing, grinding, blending).
5) Sonication, heating or boiling of samples (for instance for the extraction of nucleic acids).
6) Work with bacteriological loops: when loops charged with infectious material are placed in an ordinary bunsen burner, the material may be dispersed before it is burned and contaminate surfaces or the operator.
7) Preparation and manipulation of frozen sections (histology) when frozen material is cut, infected ice and tissue particles may be dispersed and contaminate the operator and material (even formalin-fixed tissues may still contain viable bacilli).
Need to read more
read Occupational Health Hazards in Hospitals, What Health Care Workers Should Know?
Among the laboratory techniques used for the identification and characterization of mycobacteria, the following practices are likely to increase the risk of contamination or to generate infectious aerosols producing droplet nuclei (Jensen et al., 2005):
1) Handling of containers with clinical specimens: even if this situation is unlikely to generate aerosols, it is the initial step where laboratory personnel is potentially exposed to the tubercle bacilli. It was shown that the outside of containers used for collecting clinical specimens is frequently contaminated by M. tuberculosis (6.5%) or by other airborne pathogens (15%).
2) Centrifugations: fluid may spill from centrifuge tubes or tubes may break, releasing a large amount of aerosols.
3) Pipetting: pipettes and Pasteur pipettes in particular are likely to generate bubbles which burst and form aerosols.
4) Mechanical homogenizing (vortexing, grinding, blending).
5) Sonication, heating or boiling of samples (for instance for the extraction of nucleic acids).
6) Work with bacteriological loops: when loops charged with infectious material are placed in an ordinary bunsen burner, the material may be dispersed before it is burned and contaminate surfaces or the operator.
7) Preparation and manipulation of frozen sections (histology) when frozen material is cut, infected ice and tissue particles may be dispersed and contaminate the operator and material (even formalin-fixed tissues may still contain viable bacilli).
Need to read more
read Occupational Health Hazards in Hospitals, What Health Care Workers Should Know?
Wednesday, November 9, 2011
Pyrexia of unknown origin "PUO"
Definition:
A case presented with pyrexia as a predominant clinical feature of 10 days or longer duration without an obvious cause.
It may be acute (if pyrexia persists for few days) or chronic (if pyrexia persists for 3 weeks or longer).
Causes:
I- Infective:
A) Non specific e.g:
Cryptic abscesses in liver, abdomin, pelvis and retroperitoneal or mediastinal sites.
Infective endocarditis.
Urinary tract infection.
Ear, sinus or dental infections.
Osteomyelitis.
B) Specific e.g:
Bacterial: T.B., brucellosis, typhoid F., leptospirosis (Weil’s disease), secondary syphilis.
Viral: viral hepatitis, glandular fever, yellow fever, CMV, HIV.
Rickettsial: typhus and Q fever.
Chlamydial and Bortonella: psittacosis and cat scratch fever.
Fungal: candidiasis, histoplasmosis, cryptococcosis and aspergillosis.
Protozoal: malaria, amaebiasis, toxoplasmosis, trypanosomiasis and leishmaniasis.
Helminthic: filariasis and fasciola.
II- Non-infective:
Haematological: e.g leukemia, purpura, haemolytic anaemia and lymphoma.
Autoimmune and collagen: e.g rheumatic fever, rheumatoid arthritis, SLE, polyarteritis nodosa, dermatomyositis and ulcerative colitis.
Endocrine: e.g thyrotoxicosis and familial mediteranean fever.
Malignancy: sarcoma, carcinoma, hepatoma and hypernephroma.
Miscellaneous:
Liver cirrhosis and alcoholic hepatitis.
Gout (rare).
Granulomas e.g sarcoidosis, Crohn's disease.
Drug reaction.
CNS abnormalities e.g infiltration of heat regulating center in hypothalamus by neoplasm or granuloma (rare).
Malingering.
Laboratory Diagnosis
Haematological:
Hb for anaemia.
Platelets for purpura.
WBCs: total and differential count.
Neutrophilia in pyrogenic infection.
Neutropenia in malaria; typhoid; leishmaniasis and SLE.
Lymphocytosis in viral infection, typhoid and brucellosis.
Monocytosis in TB; atypical monocytes in IMN.
Blast cells in leukemia.
Thin and thick blood film in malaria; filaria; trypanosomiasis.
ESR: > 100 mm/h in T.B; collagen and malignancy.
Microbiological:
Blood culture for typhoid, brucellosis, leptospirosis and infective endocarditis.
Urine and stool culture for UTI, gastrointestinal infections, salmonellosis, brucellosis, leptospirosis. In sterile pyuria: T.B. of genitourinary tract is suspected.
Throat swab if rheumatic fever is suspected, negative culture not exclude rheumatic fever.
Bone marrow culture for typhoid, brucellosis, T.B.
Serology:
Paired serum samples are required to look for rising antibody titer four folds. Occasionally, a single high titer maybe suggestive of recent infection e.g IgM for toxoplasma.
Some serological tests for diagnosing PUO:
Widal test for typhoid (diagnostic titre > 1/80).
Brucella agglutination and CFT (diagnostic titre >1/80).
ASO titre for rheumatic fever (diagnostic titre > 250 Todds U/ml).
Latex co-agglutination to detect Ag as Streptococcal, Staph. species, Neisseria, Candida and Rota viruses.
ELISA techniques for detection of microbial antigens e.g. Chlamydial Ag, HB Ag & HIV Ag and microbial antibodies e.g. CMV Ab, HBAb and T.B. (IgA, IgG, IgM).
Fluorescent treponemal antibody, fluorescent amaebic antibody and fluorescent leishmanial antibody test.
PCR technique for HCV-RNA, HBV-DNA, T.B-DNA, CMV & HSV.
3) Biochemical:
Liver function tests.
Thyroid function tests.
Alpha Feto Protein (AFP) for hepatoma.
Uric acid for gout.
C) Biopsy:
Bone marrow, lymph nodes, liver and transbronchial lung biopsies for culture and cytology.
D) Skin tests: e.g
Mantoux test for TB.
Kveim test for sarcoidosis.
Histoplasmin test for Histoplasmosis.
Frei test for Chlamydia (lymphogranuloma venereum).
Need to Read more
Lctures on applied clinical microbiology
A case presented with pyrexia as a predominant clinical feature of 10 days or longer duration without an obvious cause.
It may be acute (if pyrexia persists for few days) or chronic (if pyrexia persists for 3 weeks or longer).
Causes:
I- Infective:
A) Non specific e.g:
Cryptic abscesses in liver, abdomin, pelvis and retroperitoneal or mediastinal sites.
Infective endocarditis.
Urinary tract infection.
Ear, sinus or dental infections.
Osteomyelitis.
B) Specific e.g:
Bacterial: T.B., brucellosis, typhoid F., leptospirosis (Weil’s disease), secondary syphilis.
Viral: viral hepatitis, glandular fever, yellow fever, CMV, HIV.
Rickettsial: typhus and Q fever.
Chlamydial and Bortonella: psittacosis and cat scratch fever.
Fungal: candidiasis, histoplasmosis, cryptococcosis and aspergillosis.
Protozoal: malaria, amaebiasis, toxoplasmosis, trypanosomiasis and leishmaniasis.
Helminthic: filariasis and fasciola.
II- Non-infective:
Haematological: e.g leukemia, purpura, haemolytic anaemia and lymphoma.
Autoimmune and collagen: e.g rheumatic fever, rheumatoid arthritis, SLE, polyarteritis nodosa, dermatomyositis and ulcerative colitis.
Endocrine: e.g thyrotoxicosis and familial mediteranean fever.
Malignancy: sarcoma, carcinoma, hepatoma and hypernephroma.
Miscellaneous:
Liver cirrhosis and alcoholic hepatitis.
Gout (rare).
Granulomas e.g sarcoidosis, Crohn's disease.
Drug reaction.
CNS abnormalities e.g infiltration of heat regulating center in hypothalamus by neoplasm or granuloma (rare).
Malingering.
Laboratory Diagnosis
Haematological:
Hb for anaemia.
Platelets for purpura.
WBCs: total and differential count.
Neutrophilia in pyrogenic infection.
Neutropenia in malaria; typhoid; leishmaniasis and SLE.
Lymphocytosis in viral infection, typhoid and brucellosis.
Monocytosis in TB; atypical monocytes in IMN.
Blast cells in leukemia.
Thin and thick blood film in malaria; filaria; trypanosomiasis.
ESR: > 100 mm/h in T.B; collagen and malignancy.
Microbiological:
Blood culture for typhoid, brucellosis, leptospirosis and infective endocarditis.
Urine and stool culture for UTI, gastrointestinal infections, salmonellosis, brucellosis, leptospirosis. In sterile pyuria: T.B. of genitourinary tract is suspected.
Throat swab if rheumatic fever is suspected, negative culture not exclude rheumatic fever.
Bone marrow culture for typhoid, brucellosis, T.B.
Serology:
Paired serum samples are required to look for rising antibody titer four folds. Occasionally, a single high titer maybe suggestive of recent infection e.g IgM for toxoplasma.
Some serological tests for diagnosing PUO:
Widal test for typhoid (diagnostic titre > 1/80).
Brucella agglutination and CFT (diagnostic titre >1/80).
ASO titre for rheumatic fever (diagnostic titre > 250 Todds U/ml).
Latex co-agglutination to detect Ag as Streptococcal, Staph. species, Neisseria, Candida and Rota viruses.
ELISA techniques for detection of microbial antigens e.g. Chlamydial Ag, HB Ag & HIV Ag and microbial antibodies e.g. CMV Ab, HBAb and T.B. (IgA, IgG, IgM).
Fluorescent treponemal antibody, fluorescent amaebic antibody and fluorescent leishmanial antibody test.
PCR technique for HCV-RNA, HBV-DNA, T.B-DNA, CMV & HSV.
3) Biochemical:
Liver function tests.
Thyroid function tests.
Alpha Feto Protein (AFP) for hepatoma.
Uric acid for gout.
C) Biopsy:
Bone marrow, lymph nodes, liver and transbronchial lung biopsies for culture and cytology.
D) Skin tests: e.g
Mantoux test for TB.
Kveim test for sarcoidosis.
Histoplasmin test for Histoplasmosis.
Frei test for Chlamydia (lymphogranuloma venereum).
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Lctures on applied clinical microbiology
Tuesday, November 8, 2011
TNFα and IFNγ, EARLY KINETICS DURING FEVER IN PATIENTS WITH HAEMATOLOGICAL MALIGNANCIES AND NEUTROPENIA
Patients with hematological malignancies often experience long periods of profound neutropenia and in 20 - 40% of febrile episodes bacteria grow in blood culture. An altered cytokine response to bacterial infections may be anticipated in those patients.
The aim of this study is to define the levels of early TNFα and IFNγ release in febrile neutropenic patients and relate this information to blood culture results and clinical findings. In this work serum concentrations of TNFα and lFNγ were studied in 31 patients with haematological malignancies presented with fever. Blood samples were collected for TNF α and IFNγ analysis at time 0 and after 24 hours of start of fever. Blood cultures were performed for all patients at time 0. Increased levels of TNFα and IFNγ were detected at start of fever with peak values for both cytokines after 24 hours for Gram negative bacilli (Gm-ve) infections (P = 0.0) for both cytokines compared to 0 time levels). In neutropenic patients, there was a highly significant rise in both TNF α and IFNγ levels after 24 hrs (P= 0.007 & 0.001 respectively) compared to 0 time levels. There were also significant rise in both cytokines levels in cases with septic shock (P = 0.02 for TNF α and P = 0.04 for IFNγ) compared to 0 time levels.
TNF α and IFNγ can be used as early diagnostic tools in neutropenic patients with Gm-ve bacteraemia who may develop septic shock before results of blood cultures are available and this may have important therapeutic implications.
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Innate Immune Response to Pathogens and Recent Advances in Microbiology Researches
The aim of this study is to define the levels of early TNFα and IFNγ release in febrile neutropenic patients and relate this information to blood culture results and clinical findings. In this work serum concentrations of TNFα and lFNγ were studied in 31 patients with haematological malignancies presented with fever. Blood samples were collected for TNF α and IFNγ analysis at time 0 and after 24 hours of start of fever. Blood cultures were performed for all patients at time 0. Increased levels of TNFα and IFNγ were detected at start of fever with peak values for both cytokines after 24 hours for Gram negative bacilli (Gm-ve) infections (P = 0.0) for both cytokines compared to 0 time levels). In neutropenic patients, there was a highly significant rise in both TNF α and IFNγ levels after 24 hrs (P= 0.007 & 0.001 respectively) compared to 0 time levels. There were also significant rise in both cytokines levels in cases with septic shock (P = 0.02 for TNF α and P = 0.04 for IFNγ) compared to 0 time levels.
TNF α and IFNγ can be used as early diagnostic tools in neutropenic patients with Gm-ve bacteraemia who may develop septic shock before results of blood cultures are available and this may have important therapeutic implications.
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Innate Immune Response to Pathogens and Recent Advances in Microbiology Researches
Monday, November 7, 2011
Occupational Health Hazards in Hospitals, What Health Care Workers Should Know?
Occupational hazards are classified in four categories, including biological and infectious, chemical, physical and psychosocial hazards (McDiarmid, 2006):
1. Biological and infectious hazards: They include infectious and biological agents, such as bacteria, viruses, fungi, or parasites.
2. Chemical hazards: They include various forms of chemicals that are potentially toxic or irritating to the body system, including medications, solutions and gases.
3. Physical hazards: They include radiation, electricity, extreme temperatures, noise and musculoskeletal disorders.
4. Psychosocial hazards: They include factors that create or potentiate stress, emotional strain or interpersonal problems.
Chemical hazards:
There are multiple chemical agents existing in the hospital work environment. The effect of exposure depends on type and dose of the chemical, frequency and duration of exposure, work practices and the individual's susceptibility. Toxic chemicals in use in hospitals include (McDiarmid, 2006):
-Hazardous drugs requiring preparation prior to parenteral administration to the patients.
-Chemical sterilizers, in particular gluteraldehyde and ethylene oxide used for the sterilization of endoscopes and other equipment that cannot be steam sterilized.
-Tissue preservatives such as formaldehyde used to store and preserve body tissue prior to histopathology.
-Anesthetic gases in the operating theatre.
-Occupational exposure to latex allergens.
-Cleaning agents.
Radiation
Ionizing radiation is used in diagnostic procedures such as x-ray, fluoroscopy angiography and in treatment using radioactive implantations or injections. Non-ionizing radiation includes microwaves and magnetic fields used in diagnostic magnetic resonance imaging. Workers at risk of exposure are radiologists, radiologic and x-ray technician, dental assistants, dentists, physicians, nurses and nuclear medicine staff (Donaldson, 2007).
Cumulative and long-term health effects of exposure to ionizing radiation include cataracts, adverse reproductive outcomes and subclinical genetic changes, leukemia, breast and skin (basal cell) cancers. Exposure to non ionizing radiation leads to skin and eyes burns. Neurological, behavioral and immunological effects also may occur (Chodick et al., 2008).
Noise, extreme temperature and electricity:
Excessive noise and heat are commonly found in kitchens, laundries and boiler rooms. Permanent hearing loss can result from long term exposure to noise in excess of 80 decibels. At lower levels, noise from equipment, alarms, conversation and other sources can impede communication and interfere with concentration. Cold, heat and sunlight are hazards for grounds and building maintenance personnel. Skin burns can result from exposure to hot surfaces, hot liquids or from exposure to excessive sunlight. Cold temperatures can produce frostbite or a dangerous generalized cooling of the body (hypothermia). Human exposure to electrical hazards can result in burns and gaseous embolism (Donaldson, 2007).
Violence:
Workplace violence ranges from offensive or threatening language to homicide. The national institute for occupational safety and health (NIOSH) defines workplace violence as violent acts (including physical assaults and threats of assaults) directed toward persons at work or on duty. Although anyone working in a hospital may become a victim of violence, nurses and aides who have the most direct contact with patients are at higher risk. Other hospital personnel at increased risk of violence include emergency response personnel, hospital safety officers and all healthcare providers (McPhaul and Lipscomb 2004).
Violence may occur anywhere in the hospital, but it is most frequent in psychiatric wards, emergency rooms, waiting rooms and geriatric units. The effects of violence can range in intensity and include physical injuries, temporary and permanent physical disability, psychological trauma and death. Violence may also have negative organizational outcomes such as low worker morale, increased job stress, increased worker turnover, reduced trust of management and a hostile working environment (Gerberich et al., 2005).
Factors that increase violence in work are working directly with volatile people, working when understaffed especially during meal times and visiting hours, long waits for overcrowded service, uncomfortable waiting rooms, working alone, poor environmental design, inadequate security , lack of staff training and policies for preventing and managing crises with potentially volatile patients drug and alcohol abuse (McPhaul and Lipscomb 2004).
Musculoskeletal disorders:
Work related MSD are defined as an injury of the muscles, tendons, ligaments, nerves, joints, cartilage, bones and blood vessels in the extremities or back. They are caused or aggravated by manual handling work tasks such as lifting, pushing, pulling and working in postures with very repetitive or static forceful exertions (Smith et al., 2006).
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Occupational Health Hazards in Hospitals, What Health Care Workers Should Know?
http://www.amazon.com/dp/B005MJAB9O
1. Biological and infectious hazards: They include infectious and biological agents, such as bacteria, viruses, fungi, or parasites.
2. Chemical hazards: They include various forms of chemicals that are potentially toxic or irritating to the body system, including medications, solutions and gases.
3. Physical hazards: They include radiation, electricity, extreme temperatures, noise and musculoskeletal disorders.
4. Psychosocial hazards: They include factors that create or potentiate stress, emotional strain or interpersonal problems.
Chemical hazards:
There are multiple chemical agents existing in the hospital work environment. The effect of exposure depends on type and dose of the chemical, frequency and duration of exposure, work practices and the individual's susceptibility. Toxic chemicals in use in hospitals include (McDiarmid, 2006):
-Hazardous drugs requiring preparation prior to parenteral administration to the patients.
-Chemical sterilizers, in particular gluteraldehyde and ethylene oxide used for the sterilization of endoscopes and other equipment that cannot be steam sterilized.
-Tissue preservatives such as formaldehyde used to store and preserve body tissue prior to histopathology.
-Anesthetic gases in the operating theatre.
-Occupational exposure to latex allergens.
-Cleaning agents.
Radiation
Ionizing radiation is used in diagnostic procedures such as x-ray, fluoroscopy angiography and in treatment using radioactive implantations or injections. Non-ionizing radiation includes microwaves and magnetic fields used in diagnostic magnetic resonance imaging. Workers at risk of exposure are radiologists, radiologic and x-ray technician, dental assistants, dentists, physicians, nurses and nuclear medicine staff (Donaldson, 2007).
Cumulative and long-term health effects of exposure to ionizing radiation include cataracts, adverse reproductive outcomes and subclinical genetic changes, leukemia, breast and skin (basal cell) cancers. Exposure to non ionizing radiation leads to skin and eyes burns. Neurological, behavioral and immunological effects also may occur (Chodick et al., 2008).
Noise, extreme temperature and electricity:
Excessive noise and heat are commonly found in kitchens, laundries and boiler rooms. Permanent hearing loss can result from long term exposure to noise in excess of 80 decibels. At lower levels, noise from equipment, alarms, conversation and other sources can impede communication and interfere with concentration. Cold, heat and sunlight are hazards for grounds and building maintenance personnel. Skin burns can result from exposure to hot surfaces, hot liquids or from exposure to excessive sunlight. Cold temperatures can produce frostbite or a dangerous generalized cooling of the body (hypothermia). Human exposure to electrical hazards can result in burns and gaseous embolism (Donaldson, 2007).
Violence:
Workplace violence ranges from offensive or threatening language to homicide. The national institute for occupational safety and health (NIOSH) defines workplace violence as violent acts (including physical assaults and threats of assaults) directed toward persons at work or on duty. Although anyone working in a hospital may become a victim of violence, nurses and aides who have the most direct contact with patients are at higher risk. Other hospital personnel at increased risk of violence include emergency response personnel, hospital safety officers and all healthcare providers (McPhaul and Lipscomb 2004).
Violence may occur anywhere in the hospital, but it is most frequent in psychiatric wards, emergency rooms, waiting rooms and geriatric units. The effects of violence can range in intensity and include physical injuries, temporary and permanent physical disability, psychological trauma and death. Violence may also have negative organizational outcomes such as low worker morale, increased job stress, increased worker turnover, reduced trust of management and a hostile working environment (Gerberich et al., 2005).
Factors that increase violence in work are working directly with volatile people, working when understaffed especially during meal times and visiting hours, long waits for overcrowded service, uncomfortable waiting rooms, working alone, poor environmental design, inadequate security , lack of staff training and policies for preventing and managing crises with potentially volatile patients drug and alcohol abuse (McPhaul and Lipscomb 2004).
Musculoskeletal disorders:
Work related MSD are defined as an injury of the muscles, tendons, ligaments, nerves, joints, cartilage, bones and blood vessels in the extremities or back. They are caused or aggravated by manual handling work tasks such as lifting, pushing, pulling and working in postures with very repetitive or static forceful exertions (Smith et al., 2006).
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Occupational Health Hazards in Hospitals, What Health Care Workers Should Know?
http://www.amazon.com/dp/B005MJAB9O
Study of Interleukin 12-Gamma interferon Axis and Natural Killer Cells in Acute Viral hepatitis
Innate immune response toward viral hepatitis plays an important defense. Nevertheless little data are known regarding the difference of the immune response according to the type of viral pathogen.
In the present study we studied natural killer cells subsets in patients with different types of hepatitis viruses (A,B,C) combined with study of interleukin 12 (IL12) gamma interferon (IFN-γ) axis in those patients.
The study was carried out on eighty six patients with acute viral hepatitis in addition to twenty healthy subjects as control. Blood samples were subjected to study of natural killer cells counts by flow cytometry. Furthermore, serum IL12 and IFN- γ were determined by enzyme linked immunosorbent assay.
Natural killer counts had statistically significant increase among patients compared to control, P=0.036. The greatest counts were among patients with acute viral hepatitis B (25.1± 4.4) and acute viral hepatitis A (24.7± 3.5). The lowest mean was among patients with acute hepatitis C (18.6± 6.8). Furthermore, there was statistically significant difference among patients and control for both IL12 and IFN γ - (P=0.0001, P=0.022 respectively). The highest levels were found in patients with acute viral hepatitis A for IL12 and IFN- (731.9± 403.7pg/ml, 0.7± 0.0205pg/ml respectively). The lowest levels were found among patients with hepatitis B or IL12nd IFN- γ(41.2± 39.5 pg/ml, 0.1± 0.04 pg/ml, respectively) and patients with hepatitis C orIL12 and IFN- (70.0± 19.7 pg/ml, 0.12± 0.05pg/ml, respectively).
The present study highlights innate immune responses to acute viral hepatitis infections represented by IL12 and interferon gamma axis associated with natural killer. Altogether these observations are in favor of the activation of innate immune responses during acute hepatits viral infections what ever its type. Neverthless the degree of response differs according to type of infecting viruses, being extensive in hepatits A virus thought to be associated with mild form of acute hepatitis. The least response is associated with acute viral hepatitis C infection.
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Innate Immune Response to Pathogens and Recent Advances in Microbiology Researches
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In the present study we studied natural killer cells subsets in patients with different types of hepatitis viruses (A,B,C) combined with study of interleukin 12 (IL12) gamma interferon (IFN-γ) axis in those patients.
The study was carried out on eighty six patients with acute viral hepatitis in addition to twenty healthy subjects as control. Blood samples were subjected to study of natural killer cells counts by flow cytometry. Furthermore, serum IL12 and IFN- γ were determined by enzyme linked immunosorbent assay.
Natural killer counts had statistically significant increase among patients compared to control, P=0.036. The greatest counts were among patients with acute viral hepatitis B (25.1± 4.4) and acute viral hepatitis A (24.7± 3.5). The lowest mean was among patients with acute hepatitis C (18.6± 6.8). Furthermore, there was statistically significant difference among patients and control for both IL12 and IFN γ - (P=0.0001, P=0.022 respectively). The highest levels were found in patients with acute viral hepatitis A for IL12 and IFN- (731.9± 403.7pg/ml, 0.7± 0.0205pg/ml respectively). The lowest levels were found among patients with hepatitis B or IL12nd IFN- γ(41.2± 39.5 pg/ml, 0.1± 0.04 pg/ml, respectively) and patients with hepatitis C orIL12 and IFN- (70.0± 19.7 pg/ml, 0.12± 0.05pg/ml, respectively).
The present study highlights innate immune responses to acute viral hepatitis infections represented by IL12 and interferon gamma axis associated with natural killer. Altogether these observations are in favor of the activation of innate immune responses during acute hepatits viral infections what ever its type. Neverthless the degree of response differs according to type of infecting viruses, being extensive in hepatits A virus thought to be associated with mild form of acute hepatitis. The least response is associated with acute viral hepatitis C infection.
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Innate Immune Response to Pathogens and Recent Advances in Microbiology Researches
http://www.amazon.com/dp/B005YKC75C
Genetic and phenotypic characterization of drug-resistant Mycobacterium tuberculosis isolates
The reemergence of Mycobacterium tuberculosis with increasing numbers of multi-drug resistant (MDR) strains has increased the need for rapid diagnostic methods.
Molecular bases of drug resistance have been identified for all of the main antituberculous drugs, and drug resistance results from changes in several target genes, some of which are still undefined. Drug resistance in M. tuberculosis is due to the acquisition of mutations in chromosomally encoded genes and the generation of multidrug resistance is a consequence of serial accumulation of mutations primarily due to inadequate therapy. Several studies have shown that resistance to isoniazid (INTI) is due to mutaions in kat G gene. The rpo B gene, which encodes the subunit of RNA polymerase, harbors a mutation in an 81 bp region in about 95% of rifampicin (RIF) reistant M. tuberculosis strains recovered globally. Streptomycin (STR) resistance is due to mutations in rrs and rpsl genes which encodes 16S SrRNA and ribosomal protein S12 respectively. Approximately 65% of clinical isolates resistant to ethambutol have a mutation in the embB gene.
Several susceptibility phenotypes methods have been developed. Among them were radiometric systems such as the BACTEC460 TB system reduces the test time considerably, but they still labor intensive, expensive and require manipulation of radioactive substances.
Rapid promising approach to determine drug resistant strains especially for rifampicin (RIF) is the use of mycobacteriophage. A number of low-cost colorimetric AST assays, such as the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, the Alamar blue assay, and an assay based on microscopic detection of cord-like growth by M. tuberculosis, have been described. However, these tests have limitations; mycobacteria other than M. tuberculosis can produce cord factor, and INH can interfere with the formazan production in the MTT assay and give rise to false resistant results. Moreover, the use of a liquid medium in a microtiter plate format in these tests may be disadvantageous not only as a biohazard but also due to possible Contamination between wells.
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Mycobacterium Tuberculosis, Current status in Rapid Laboratory Diagnosis
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Molecular bases of drug resistance have been identified for all of the main antituberculous drugs, and drug resistance results from changes in several target genes, some of which are still undefined. Drug resistance in M. tuberculosis is due to the acquisition of mutations in chromosomally encoded genes and the generation of multidrug resistance is a consequence of serial accumulation of mutations primarily due to inadequate therapy. Several studies have shown that resistance to isoniazid (INTI) is due to mutaions in kat G gene. The rpo B gene, which encodes the subunit of RNA polymerase, harbors a mutation in an 81 bp region in about 95% of rifampicin (RIF) reistant M. tuberculosis strains recovered globally. Streptomycin (STR) resistance is due to mutations in rrs and rpsl genes which encodes 16S SrRNA and ribosomal protein S12 respectively. Approximately 65% of clinical isolates resistant to ethambutol have a mutation in the embB gene.
Several susceptibility phenotypes methods have been developed. Among them were radiometric systems such as the BACTEC460 TB system reduces the test time considerably, but they still labor intensive, expensive and require manipulation of radioactive substances.
Rapid promising approach to determine drug resistant strains especially for rifampicin (RIF) is the use of mycobacteriophage. A number of low-cost colorimetric AST assays, such as the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, the Alamar blue assay, and an assay based on microscopic detection of cord-like growth by M. tuberculosis, have been described. However, these tests have limitations; mycobacteria other than M. tuberculosis can produce cord factor, and INH can interfere with the formazan production in the MTT assay and give rise to false resistant results. Moreover, the use of a liquid medium in a microtiter plate format in these tests may be disadvantageous not only as a biohazard but also due to possible Contamination between wells.
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Mycobacterium Tuberculosis, Current status in Rapid Laboratory Diagnosis
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Sunday, November 6, 2011
Natural Killer Activity and Mycobacterium tuberculosis Infection
There is recent increase in tuberculosis with multidrug resistant strains. On the other hand little information is available about the role of natural killer cells (N.K) in the immunity to tuberculosis and antituberculous sensitivity.
In this work 25 chest patients and 6 controls were studied for N.K activity by release of Cr from erythroleukaemia cell line. T.B diagnosis was performed by culture and sensitivity with Bactec system . specific serology (1gA , 1gG, 1gM) and tuberculin test.
All patients have typical mycobacteria tuberculosis with sensitivity 72% to isoniazid, 56% to rifampicin, 36 % to ethambutol, 32% to streptomycin and 16 % were resistant to all drugs . Tuberculin was positive in 56%, 1gG and 1gM in 16 % and 1gA in 8%. There was significant decrease (P < .0001) in N.K activity in patients compared to control. The results suggest that the N.K cells play role in defense mechanism to T.B.
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Innate Immune Response to Pathogens and
Recent Advances in Microbiology Researches
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In this work 25 chest patients and 6 controls were studied for N.K activity by release of Cr from erythroleukaemia cell line. T.B diagnosis was performed by culture and sensitivity with Bactec system . specific serology (1gA , 1gG, 1gM) and tuberculin test.
All patients have typical mycobacteria tuberculosis with sensitivity 72% to isoniazid, 56% to rifampicin, 36 % to ethambutol, 32% to streptomycin and 16 % were resistant to all drugs . Tuberculin was positive in 56%, 1gG and 1gM in 16 % and 1gA in 8%. There was significant decrease (P < .0001) in N.K activity in patients compared to control. The results suggest that the N.K cells play role in defense mechanism to T.B.
Need to read more
Innate Immune Response to Pathogens and
Recent Advances in Microbiology Researches
http://www.amazon.com/dp/B005YKC75C
Friday, November 4, 2011
Interleukin 6 response in Children with Echerichia coli associated Urinary tract Infection, One centre Study.
Background: Urinary tract infection (UTI) is one of the common pediatric health problem.
The aim of the present study was to evaluate urinary tract infections in children in relations to virulence factors of Escherichia coli and to innate immune response with respect to IL6.
Methods: This investigation has been performed on a number of 2722 children their ages ranged from 1 to 12 years with a history of urinary tract infections (UTI) undergoing clinical manifestations presented to Pediatric University Hospital in Mansoura, Egypt. Urine samples were subjected to full microbiological assessment and measurement of interleukin 6 was performed for samples associated with Escherichia coli (E.coli). Furthermore, fimbriae genotypes papG were determined by multiplex PCR.
Results: Escherichia coli was the commonest isolate. For fimbria papG genotypes study, the commonest alleles were combined II and III followed by I and III. The interesting finding was the statistically significant association of allele II with multidrug resistant E.coli (51%). IL6 had significant positive correlations with both bacterial counts, total leucocytic counts in urine and. the presence of papG allele I and allele III (177pg/ml ±132, P=0.0001
Conclusions: There was high prevalence of UTI among patients in our study. The commonest pathogen was Escherichia coli. The results demonstrate that infections of the urinary tract activate IL-6 response in children and that the magnitude of the IL-6 response is influenced by the properties of the infecting strain. The severity of the infection represented by resistant to antibiotics and high urinary IL6 was associated with fimbria genotypes II and III. Urinary IL6 level can be used as a rapid biomarker for assessment of the severity of UTI in children.
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The aim of the present study was to evaluate urinary tract infections in children in relations to virulence factors of Escherichia coli and to innate immune response with respect to IL6.
Methods: This investigation has been performed on a number of 2722 children their ages ranged from 1 to 12 years with a history of urinary tract infections (UTI) undergoing clinical manifestations presented to Pediatric University Hospital in Mansoura, Egypt. Urine samples were subjected to full microbiological assessment and measurement of interleukin 6 was performed for samples associated with Escherichia coli (E.coli). Furthermore, fimbriae genotypes papG were determined by multiplex PCR.
Results: Escherichia coli was the commonest isolate. For fimbria papG genotypes study, the commonest alleles were combined II and III followed by I and III. The interesting finding was the statistically significant association of allele II with multidrug resistant E.coli (51%). IL6 had significant positive correlations with both bacterial counts, total leucocytic counts in urine and. the presence of papG allele I and allele III (177pg/ml ±132, P=0.0001
Conclusions: There was high prevalence of UTI among patients in our study. The commonest pathogen was Escherichia coli. The results demonstrate that infections of the urinary tract activate IL-6 response in children and that the magnitude of the IL-6 response is influenced by the properties of the infecting strain. The severity of the infection represented by resistant to antibiotics and high urinary IL6 was associated with fimbria genotypes II and III. Urinary IL6 level can be used as a rapid biomarker for assessment of the severity of UTI in children.
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URINARY TRACT INFECTIONS
Samples of urine from patients with suspected infections of the urinary tract are the most numerous, e.g. 30-40%. of the different kinds of specimens received in most clinical labora¬tories. The schedule for their routine examin¬ation should therefore be carefully determined with a view to obtaining the necessary diagnostic information with the greatest possible economy of labor and resources.
The examinations generally made are the microscopical examination of a wet film of uncentrifuged urine to determine whether poly¬morphs (‘pus cells’) are present in numbers indicative of infection in the urinary tract, and the semi-quantitative culture of the urine to determine whether it contains a potentially patho¬genic bacterium in numbers sufficient to ident¬ify it as the causal infecting organism (‘significant bacteriuria’).
The chemotherapy of a proven infection may be guided by in-vitro sensitivity tests on the pathogen isolated in culture and the outcome of therapy assessed by examination of the urine at the conclusion of treatment. Follow-up examin¬ation of patients who have had urinary tract infection is advisable because a relapse may be clinically silent.
The common symptoms of urinary tract infect¬ion are urgency and frequency of micturition, with associated discomfort or pain. The commonest condition is cystitis, due to infection of the bladder with a uropathogenic bacterium, which most frequently is Escherichia coli but sometimes Staphylococcus saprophyticus or, especially in hospital-acquired infections. Klebsiella pneumoniae var. aerogenes or oxyloca, Proteus mirabilis, other coliforms and Pseudomonas aeruginosa or Streptococcus faecalis. Candida infection may occur in diabetic and immuno-compromised patients. Rarer infecting organisms include Streptococcus agulacziae, Streptococcus milleri, and other streptococci, anaerobic streptococci and Gardenella vaginalis. There has been much debate on the significance of so-called fastidious organisms in urinary tract infection.
More serious bacterial infections are acute pyelitis and pyelonephritis in which the symptoms usually include loin pain and fever and which may be accompanied by a bacteriaemia detectable by blood culture. The causative organism may be any of those that cause cystitis, but Staphylococcus aureus is responsible for some of the cases.
Patients with signs or symptoms of urinary tract infection sometimes produce samples of urine that show pus cells but do not yield a significant growth of bacteria on routine culture.
The explanation may be that the patient has been taking antibiotics prescribed on a previous, occasion. Alternatively, he may have an infection with an organism that does not grow’ on the media normally used for routine investigations. In such cases it is important to consider genitourinary tuberculosis or gonococcal infection and infection with nutrition¬ally exacting or anaerobic bacteria. But many patients with frequency and dysuria do not have a bacterial infection of the bladder, nor significant numbers of bacteria in their urine (abacterial pyuria). Their condition is known as non-bacterial urethritis or cystitis, or the urethral syndrome. The cause of which may be urethral or bladder infection with a chlam¬ydia, ureaplasma, trichomonas or virus, which often remains unrecognized.
Screening out negatives
About 70-80% of the urine specimens received in a clinical laboratory are found on full microscopical and cultural examinations to be free from evidence of infection in the urinary tract. A variety of chemical and automated methods have been tried for the detection of the negative specimens, but none has yet been generally accepted as sufficiently reliable for its purpose. Recently. it has been reported that the finding of negative results in all of three chemical tests for nitrite, blood and protein, performed by a rapid automated dip-strip method (N-Labstix. Ames), predicts the absence of bacteriuria in about half of the culture-negative specimens. which may then be discarded.
Significant bacteriuria
The specimen most easily and therefore most commonly collected is mid-stream urine (MSU).
Although the greater part of the urinary tract is devoid of a commensal flora and bladder urine ii an uninfected person is free from bacteria, a specimen of spontaneously voided urine is to be contaminated with some commensal bacteria from the urethral orifice and perineum, particu¬larly in females, even when the most careful precautions are taken to prevent such contami¬nation. As these contaminating commensals include the very bacteria, such as E. coli and S. saprophyticus, which are the commonest organ¬isms to infect the urinary tract, the simple demonstration that bacteria of one of these species are present in the sample of urine is not poof that it has been derived from an infection in the urinary tract.
Proof of a urinary tract infection requires the demonstration that the potential pathogen is present freshly voided urine in numbers greater than those likely to result from contamination from the urethral meatus and its environs . The observations suggested that this number, taken to indicate significant bacteriuria , is about 100 000/ml. In true infections, in the absence or chemo therapy , the number of the infecting bacteria is likely to be at least as great as this. Accordingly a quantitative method of culture is adopted to estimate the number of viable bacteria in the specimen.
When properly collected specimens of urine are examined, contamination accounts for less than 104 organisms/ml and usually for less than 103/ml. Counts due to contamination are variable and the colonies often of diverse species. Specimens from urinary tract infections almost always contain more than 104 organisms/ml, usually more than 105/ml and often up to 108/ml. These high counts, which are fairly constant in serial specimens from the same patient, reflect bacterial multiplication in the urine in vivo and are accepted as indicating significant bacteriuria. The growth obtained in such cases usually represents a single infecting species, though some infections with two species. e.g. E. colil and S. faecalis, are encountered.
Significant bacieriuria (count >105/ml in a carefully taken and promptly examined sample) may sometimes occur in the absence of symp¬toms and pyuria in patients who subsequently develop symptoms of urinary tract infection. e.g. in pregnancy. The detection of such asymp¬tomatic bacteriuria is of value for there is good evidence of its association with the development of pyelonephritis in some patients.
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The examinations generally made are the microscopical examination of a wet film of uncentrifuged urine to determine whether poly¬morphs (‘pus cells’) are present in numbers indicative of infection in the urinary tract, and the semi-quantitative culture of the urine to determine whether it contains a potentially patho¬genic bacterium in numbers sufficient to ident¬ify it as the causal infecting organism (‘significant bacteriuria’).
The chemotherapy of a proven infection may be guided by in-vitro sensitivity tests on the pathogen isolated in culture and the outcome of therapy assessed by examination of the urine at the conclusion of treatment. Follow-up examin¬ation of patients who have had urinary tract infection is advisable because a relapse may be clinically silent.
The common symptoms of urinary tract infect¬ion are urgency and frequency of micturition, with associated discomfort or pain. The commonest condition is cystitis, due to infection of the bladder with a uropathogenic bacterium, which most frequently is Escherichia coli but sometimes Staphylococcus saprophyticus or, especially in hospital-acquired infections. Klebsiella pneumoniae var. aerogenes or oxyloca, Proteus mirabilis, other coliforms and Pseudomonas aeruginosa or Streptococcus faecalis. Candida infection may occur in diabetic and immuno-compromised patients. Rarer infecting organisms include Streptococcus agulacziae, Streptococcus milleri, and other streptococci, anaerobic streptococci and Gardenella vaginalis. There has been much debate on the significance of so-called fastidious organisms in urinary tract infection.
More serious bacterial infections are acute pyelitis and pyelonephritis in which the symptoms usually include loin pain and fever and which may be accompanied by a bacteriaemia detectable by blood culture. The causative organism may be any of those that cause cystitis, but Staphylococcus aureus is responsible for some of the cases.
Patients with signs or symptoms of urinary tract infection sometimes produce samples of urine that show pus cells but do not yield a significant growth of bacteria on routine culture.
The explanation may be that the patient has been taking antibiotics prescribed on a previous, occasion. Alternatively, he may have an infection with an organism that does not grow’ on the media normally used for routine investigations. In such cases it is important to consider genitourinary tuberculosis or gonococcal infection and infection with nutrition¬ally exacting or anaerobic bacteria. But many patients with frequency and dysuria do not have a bacterial infection of the bladder, nor significant numbers of bacteria in their urine (abacterial pyuria). Their condition is known as non-bacterial urethritis or cystitis, or the urethral syndrome. The cause of which may be urethral or bladder infection with a chlam¬ydia, ureaplasma, trichomonas or virus, which often remains unrecognized.
Screening out negatives
About 70-80% of the urine specimens received in a clinical laboratory are found on full microscopical and cultural examinations to be free from evidence of infection in the urinary tract. A variety of chemical and automated methods have been tried for the detection of the negative specimens, but none has yet been generally accepted as sufficiently reliable for its purpose. Recently. it has been reported that the finding of negative results in all of three chemical tests for nitrite, blood and protein, performed by a rapid automated dip-strip method (N-Labstix. Ames), predicts the absence of bacteriuria in about half of the culture-negative specimens. which may then be discarded.
Significant bacteriuria
The specimen most easily and therefore most commonly collected is mid-stream urine (MSU).
Although the greater part of the urinary tract is devoid of a commensal flora and bladder urine ii an uninfected person is free from bacteria, a specimen of spontaneously voided urine is to be contaminated with some commensal bacteria from the urethral orifice and perineum, particu¬larly in females, even when the most careful precautions are taken to prevent such contami¬nation. As these contaminating commensals include the very bacteria, such as E. coli and S. saprophyticus, which are the commonest organ¬isms to infect the urinary tract, the simple demonstration that bacteria of one of these species are present in the sample of urine is not poof that it has been derived from an infection in the urinary tract.
Proof of a urinary tract infection requires the demonstration that the potential pathogen is present freshly voided urine in numbers greater than those likely to result from contamination from the urethral meatus and its environs . The observations suggested that this number, taken to indicate significant bacteriuria , is about 100 000/ml. In true infections, in the absence or chemo therapy , the number of the infecting bacteria is likely to be at least as great as this. Accordingly a quantitative method of culture is adopted to estimate the number of viable bacteria in the specimen.
When properly collected specimens of urine are examined, contamination accounts for less than 104 organisms/ml and usually for less than 103/ml. Counts due to contamination are variable and the colonies often of diverse species. Specimens from urinary tract infections almost always contain more than 104 organisms/ml, usually more than 105/ml and often up to 108/ml. These high counts, which are fairly constant in serial specimens from the same patient, reflect bacterial multiplication in the urine in vivo and are accepted as indicating significant bacteriuria. The growth obtained in such cases usually represents a single infecting species, though some infections with two species. e.g. E. colil and S. faecalis, are encountered.
Significant bacieriuria (count >105/ml in a carefully taken and promptly examined sample) may sometimes occur in the absence of symp¬toms and pyuria in patients who subsequently develop symptoms of urinary tract infection. e.g. in pregnancy. The detection of such asymp¬tomatic bacteriuria is of value for there is good evidence of its association with the development of pyelonephritis in some patients.
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