Characterization of Enterococcus faecium isolates and first report of vanB phenotype-vanA genotype incongruence in the Middle East.

We aimed to characterize the vancomycin genotype/phenotype, carriage of putative virulence genes, and genetic relatedness of Enterococcus faecium isolates in Saudi Arabia. E. faecium isolated from inpatients at our medical center were studied. Sensitivity to ampicillin, linezolid, teicoplanin, quinupristin/dalfopristin, tetracycline, and ciprofloxacin was determined. The presence of van genes and virulence genes for aggregation substance (Asa-1), enterococcal surface proteins (esp), cytolysin (cylA, cylL, cylM), gelatinase (gelE), E. faecium endocarditis antigen (EfaA( fm )), hyaluronidase (hyl), and collagen adhesion (Ace) was assessed. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE). Twenty-nine E. faecium isolates were obtained and the majority of isolates (n/N = 22/29) were from stool specimens. PFGE analysis identified eight pulsotypes (A-H) based on 80 % similarities. Isolates were represented in five major pulsotypes: type A (n = 5), type B (n = 3), type D (n = 6), type E (n = 5), and type F (n = 7). All isolates were vanA gene-positive. Thirteen isolates had vanA(+)/vanB(+) genotype. Of these, ten exhibited a vanB phenotype and three had a vanA phenotype. Eight isolates with vanA(+)/vanB(-) genotype exhibited vanB phenotype. Six of these eight isolates belonged to the same pulsotype. All isolates were positive for gelE, esp, and EfaA( fm ) genes. Five were CylA-positive and 24 had the hyl genes. Of the eight isolates harboring a combination of gelE, esp, EfaA( fm ), and hyl genes, five showed vanB phenotype-vanA genotype incongruence. This is the first report of vanB phenotype-vanA genotype incongruent E. faecium in the Middle East region. Molecular typing indicates clonal spread and high occurrence of virulence genes, especially esp genes, associated with epidemic clones.

Antibiotic therapeutic options for infections caused by drug-resistant Gram-positive cocci.

Serious infections caused by Gram-positive bacteria are currently difficult to treat because many of these pathogens are now resistant to standard antimicrobial agents. As a result of the emergence and spread of multidrug-resistant Gram-positive pathogens, new antimicrobial agents are urgently needed for clinical use. In recent years, there has been an increase in the number of drugs that have activity against these Gram-positive pathogens. Daptomycin, tigecycline, linezolid, quinupristin/dalfopristin and dalbavancin are five antimicrobial agents that are useful for the treatment of infections due to drug-resistant Gram-positive cocci. This review focuses on their mechanism of action, pharmacokinetics, spectrum of activity, clinical effectiveness, drug interaction and safety. These antimicrobial agents provide the clinician with additional treatment options among the limited therapies for resistant Gram-positive bacterial infection.

The management of Clostridium difficile infection: antibiotics, probiotics and other strategies.

Clostridium difficile-associated disease remains an important nosocomial infection associated with significant morbidity and mortality. In recent years, there has been an upward trend in the incidence of this condition with continuing high rates of recurrent disease with available treatment regimens. In this article, we review the current literature on the management of C. difficile-associated disease (CDAD). The potential role for alternative therapeutic options for the treatment of CDAD, including the use of bacteriotherapy in the form of probiotics, immunotherapy and ion-exchange resins as well as new drugs under investigation is explored. The evidence indicates a need for innovative approaches to the management of this condition. The combined use of antibiotic therapy and replacement of gut microbiota using probiotics remains promising and we suggest a multi-pronged approach in the management of this challenging infection.

Ultrastructural assessment of Plasmodium falciparum in age-fractionated thalassaemic erythrocytes.

Culture of Plasmodium falciparum in age-fractionated thalassaemic red blood cells (RBC) has shown evidence of parasite damage on light microscopy in older cells during the third culture cycle (96-144 h). In this report, parasites growing in thalassaemic trait and normal RBC were examined ultrastructurally from 96 to 144 h. All parasite stages in old thalassaemic RBC showed evidence of damage worsening with culture duration. There were cytoplasmic alterations with ribosomal damage, and parasite cytoplasm became increasingly loose and grainy, with multiple fissures. Discontinuity of the nuclear membrane with an abnormal nucleolus was seen at l20 h. Cytosomes remained normal, but damage to the food vacuole and shrunken disintegrating parasites were observed at 144 h. These changes are compatible with cellular degeneration and developmental retardation and would account for the schizont maturation arrest and reduced reinvasion rates previously reported. Increased free radicals associated with thalassaemic erythrocytes would explain these changes, further supporting the role for oxidant stress in the protective mechanism.

Probiotics: facts and myths.

In recent years there has been a significant upsurge in research on the characterisation and verification of the potential health benefits associated with the use of probiotics. In addition, the market for probiotics continues to expand exponentially as consumers (mostly healthy individuals) rely on health claims made by manufacturers to make their choices. This review appraises the available evidence for and against the health claims associated with probiotics. The use of probiotics in promoting gastrointestinal health and immunity, and their use in the prevention of urogenital infections, allergies and cancer are reviewed. Furthermore, issues surrounding the use of probiotics in healthy individuals, the safety of probiotics and regulatory concerns are addressed. There is scientific evidence that specific strains of probiotic microorganisms confer health benefits on the host and are safe for human use. However, this evidence cannot be extrapolated to other strains, as these effects are strain-specific. Probiotics have potential health benefits for conditions such as gastrointestinal infections, genitourinary infections, allergies and certain bowel disorders, all of which afflict a considerable proportion of the global population. However, considerable work is still needed to confirm these potential health benefits.

In vitro sensitivity of artemeter in Plasmodium falciparum-infected beta-thalassaemic trait erythrocytes.

Using an age-fractionated RBC model, we investigated the in vitro sensitivity of artemether in beta-thalassaemic RBC infected with the K1 and FC27 strains of Plasmodium falciparum and, to study the role of oxidant stress in modulating the sensitivity pattern, pro-oxidant (riboflavin) and antioxidant (vitamin E) agents were added to cultures in the presence of artemether. With the FC27 strain, the artemether IC50 doses in thalassaemic samples (whole blood and fractions) were significantly higher compared to equivalent normal RBC samples (P < 0.05). However, with the K1 strain, such a significant difference was not demonstrable. The addition of vitamin E reduced the antimalarial effect of artemether in both the FC27 and K1 strains (P < 0.0001). In contrast, the addition of riboflavin resulted in a significant increase in antimalarial activity (P < 0.0001). This effect of the drug combinations was not influenced by the red cell type (P < 0.0001) and there was no interaction between red cell type and drug type (P < 0.0001). These findings show that reduced sensitivity to artemether occurs in whole blood and age-fractionated beta-thalassaemic trait RBC. It appears that the RBC redox status does not influence the sensitivity to artemether.

Red cell age and susceptibility to malaria during pregnancy.

BACKGROUND: Increased susceptibility to malaria in pregnancy is well recognized, and has generally been assumed to be due to hormonal changes resulting in altered immunity. Based on previous work demonstrating enhanced parasite growth in young normal and thalassemic red blood cells, we hypothesized that in pregnancy increased malaria susceptibility may be due, in part, to the increase in the population of young red cells. METHODS: FC27 strain of Plasmodium falciparum was cultured in the red cells and sera from healthy primigravida pregnant (n=9) and non-pregnant (n=9) women. Red cells from both pregnant and non-pregnant women were each placed in three cultures containing the sera from pregnant, non-pregnant and pooled control samples. Cultures were set up in triplicate and incubated for 144 hours. Parasite development and growth were assessed by slide microscopy. RESULTS: At 96 hours the median parasite growth in cells from pregnant samples (5.7%) was significantly better than that in the non-pregnant cells (4.8%) (p=0.01). There was no significant difference in parasite growth in cultures with pregnant and non-pregnant sera. As expected, there were significant differences in parameters measuring red cell age between the cells from pregnant and nonpregnant samples: median red cell creatine (11.09 mg/dl) versus (6.90 mg/dl) (p=0.004) and median reticulocyte count (2.3%) versus (1.4%) (p=0.0006). CONCLUSIONS: These preliminary results are consistent with the hypothesis that an increased population of young red cells may contribute to increased malaria susceptibility during pregnancy.

Flow cytometric assessment of oxidant stress in age-fractionated thalassaemic trait erythrocytes and its relationship to in vitro growth of Plasmodium falciparum.

The role of oxidant stress in mediating the protection against malaria in thalassaemic red blood cells (RBC) has been hypothesized. In this study we have assessed the relationship between oxidant stress, red cell age and malarial parasite activity in thalassaemic RBC. Using a flow cytometric method to assess lipid peroxidation, we have shown that the age-related increase in sensitivity to oxidative stress previously demonstrated in normal RBC also occurs in thalassaemic RBC. Invasion and growth of Plasmodium falciparum was also shown to deteriorate with increasing RBC age. This effect was more pronounced in thalassaemic RBC with associated schizont maturation arrest and abnormal parasite morphology. In addition, there was a slight but consistent inverse correlation between sensitivity to oxidant stress and parasite activity (R = -0.43; P = 0.03 for normal RBC and R = -0.42; P = 0.01 for thalassaemic RBC). Our findings indicate an association between red cell age, oxidant stress and P. falciparum growth, providing further support for the role of oxidant stress in mediating the protective effect against malaria in thalassaemic RBC.

Meningitis in Hong Kong children, with special reference to the infrequency of haemophilus and meningococcal infection.

OBJECTIVE: To study the epidemiologic and aetiologic features of meningitis in children in Hong Kong. METHODOLOGY: A retrospective study of 85 children resident in the New Territory East region of Hong Kong admitted to a teaching Hospital because of meningitis during a 9 year period. RESULTS: Mycobacterium tuberculosis was the most common aetiological agent accounting for 13 cases (15.3%). Other bacteria accounted for 41 cases (48%); among these one fifth were caused by Haemophilus influenzae type b. The overall admission rates for tuberculous meningitis in Chinese children were 0.76/100,000 (95% CI 0.25-1.78) and 0.42/100,000 (CI 0.19-0.8) per year, respectively, for under 5 year olds and under 15 year olds. The overall annual incidence rates of bacterial meningitis other than tuberculous were 5.2/100,000 (CI 3.72-7.43) and 1.6/100,000 (CI 1.14-2.29) for Chinese children under 5 years and under 15 years, respectively. The annual incidence of H. influenzae meningitis in Chinese children under 5 years old was low at 1.1/100,000 (0.43-2.2). All five cases of meningococcal meningitis were in Vietnamese children (under 5 years of age incidence: 13.0/100,000 per year, CI 4.2-30.3). There were no cases of meningococcal meningitis in Chinese children during the 9 year period. CONCLUSION: M. tuberculosis was the most common aetiological agent of meningitis in Hong Kong children. The incidence of haemophilus or meningococcal meningitis was very low.

Invasion and growth of Plasmodium falciparum is inhibited in fractionated thalassaemic erythrocytes.

Epidemiological and clinical studies have indicated that the thalassaemias may confer protection against malaria. The study reported here investigated this protective effect in vitro, using a new approach which controls for the potential effect of red cell size and age on the virulence of the parasite. A Percoll density gradient method was used to separate alpha- and beta-thalassaemic trait, haemoglobin H and normal red blood cells (RBC) into fractions of different density. Correlations between RBC density, age and size in fractions of all genotypes were established using red cell creatine as an index of cell age. The development of Plasmodium falciparum over 3 erythrocytic cycles (144 h) in whole blood as well as fractionated samples was monitored by slide microscopy and flow cytometry. A significantly reduced rate of parasite invasion and growth was demonstrated in RBC from all thalassaemic genotypes tested. Poor reinvasion rates were noted in the second and third cycles. Increased duration of culture and red cell age also had a greater negative impact on parasite growth in thalassaemic RBC. This poor growth rate was also associated with the arrest of parasite growth at the schizont stage (schizont maturation arrest) and the accumulation of abnormal, trophozoite/schizont stage parasites in the older thalassaemic RBC fractions. These findings suggest a defect in the number and viability of merozoites generated by parasites growing in thalassaemic RBC. Age related factors such as oxidant stress may play a key role in mediating this kind of protective mechanism and deserve further investigation.

Thalassaemia trait, red blood cell age and oxidant stress: effects on Plasmodium falciparum growth and sensitivity to artemisinin.

Knowledge of innate mechanisms of protection against malaria could be used to bolster the existing limited treatments. Oxidant stress may play a role in the protective mechanism and the effect of red blood cell (RBC) age has recently been recognized. This study investigated the role of oxidant stress in the protection against malaria in thalassaemic trait RBC (alpha and beta) using an experimental approach which controlled for cell age. 'Young', 'intermediate' and 'old' RBC obtained by Percoll fractionation and whole blood were used to set up malaria cultures. Antioxidants (vitamin E and dithiothreitol) and pro-oxidants (riboflavin, menadione and artemisinin) were added to modulate oxidant stress effect. Antioxidants improved parasite growth. The degree of improvement was significantly greater with increasing RBC age (P < 0.0001), and relatively greater in thalassaemic RBC (P < 0.0001). Pro-oxidants had a parasiticidal effect. With the exception of the 'old' RBC fraction, the median inhibitory concentration (IC50) for riboflavin and menadione was significantly higher in normal RBC. In contrast, the IC50 for artemisinin was significantly higher in 'old' thalassaemic RBC but was similar in the 'young' and 'intermediate' fractions and whole blood. These findings suggest that oxidant stress plays a role in mediating the protection against malaria in thalassaemic RBC. Vitamin E and other antioxidant supplementation could feasibly exacerbate clinical malaria. Conversely, pro-oxidant agents could act as useful adjuvants to therapy. It is important to confirm the reduced sensitivity to artemisinin in 'old' thalassaemic trait RBC, as such an effect may promote selective pressure for the emergence of resistant parasite strains with widespread use of artemisinin.