Indirect hemagglutination antibodies against Burkholderia pseudomallei in normal blood donors and suspected cases of melioidosis in Malaysia

Interpretation of the indirect hemagglutination test (IHA) for melioidosis in endemic areas is difficult because of the presence of antibodies in apparently healthy individuals. Fifty-three out of 200 healthy blood donors in Malaysia showed positive antibody titers (> or = 1 : 40) against Burkholderia pseudomallei. Seven percent had an IHA titer of 1 : 40, 11% had an IHA titer of 1 : 80 while 8.5% had a titer > or = 1 : 160. Out of 258 sera sent for melioidosis serology, 7% of the patients had an IHA titer of 1 : 40, 9% had an IHA titer of 1 : 80 while 20% had an IHA titer of > or = 1 : 160. If a titer of > or = 1 : 80 is taken as cut off point for positivity, 29% of the patients had positive melioidosis serology. Increasing the positivity threshold may jeopardize the sensitivity of the test. A more specific and sensitive test is needed.     

Molecular procedure for rapid detection of Burkholderia mallei and Burkholderia pseudomallei

A PCR procedure for the discrimination of Burkholderia mallei and Burkholderia pseudomallei was developed. It is based on the nucleotide difference T 2143 C (T versus C at position 2143) between B. mallei and B. pseudomallei detected in the 23S rDNA sequences. In comparison with conventional methods the procedure allows more rapid identification at reduced risk for infection of laboratory personnel.     

Stability of vancomycin-resistant enterococcal genotypes isolated from long-term-colonized patients

The Eph-related receptor tyrosine kinases constitute a large family of receptors with most members displaying specific expression patterns in the developing embryo. Ligands for Eph receptor tyrosine kinases, recently renamed ephrins, comprise a family of at least 8 membrane-bound members that display promiscuous binding to Eph receptors. Here we report the characterization of a human cDNA clone with high homology to the gene encoding the murine ephrin-A2 ligand. The human gene encodes a single 2.4-kb mRNA with a restricted and developmentally-regulated tissue distribution pattern. In the fetus, ephrin-A2 mRNA is expressed in brain and intestine, whereas in the adult, high levels of ephrin-A2 mRNA are detectable in lung and intestine. Using PCR-based screening of genomic DNA from human x rodent hybrid cell lines, the gene encoding ephrin-A2 (EFNA2) was assigned to chromosome 19. Fluorescence in situ hybridization to metaphase chromosome preparations refined this localization to band p13.3.     

Purification and characterization of a cytotoxic exolipid of Burkholderia pseudomallei

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease, which is increasingly recognized as an important public health problem in various tropical regions. This study describes the identification and characterization of a heat-stable extracellular toxin of B. pseudomallei. After cultivation of B. pseudomallei in liquid media, the heated cell-free supernatant was concentrated by ultrafiltration. The concentrate exhibited a cytotoxic and hemolytic activity which showed remarkable resistance against alkaline and acidic treatments. For further purification, reversed-phase chromatography using a fast-performance liquid chromatography system was performed. After elution with an acetonitrile gradient, a single cytotoxic and hemolytic peak was detected. Structural characterization of the toxin was performed by a combination of mass spectrometric and nuclear magnetic resonance spectroscopic techniques. A highly purified glycolipid, 2-O-alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-beta-hydroxytetradec anoyl-beta-hydroxytetradecanoate (Rha-Rha-C14-C14), with a molecular mass of 762 Da was identified. The purified exolipid showed a time- and dose-dependent cytotoxic effect on phagocytic (HL60) and nonphagocytic (HeLa) cell lines. In addition, a time- and dose-dependent hemolysis of erythrocytes from various species was observed. The toxin structure makes a detergentlike action most probable. Interestingly, the cytotoxic and hemolytic activities of the glycolipid could be neutralized by albumin. Future studies will concentrate on the role of this exolipid as a virulence factor in the pathogenesis of melioidosis.     

Isolation and identification of Burkholderia pseudomallei from soil using selective culture techniques and the polymerase chain reaction

An environmental soil survey to detect Burkholderia pseudomallei was performed during the dry and wet seasons in Darwin, Northern Territory, Australia. Soil was sampled at regular intervals during a 15-month period at different depths from areas which were representative of the local, soil environment. Selective culture techniques using Ashdown's and Galimand and Dodin's methods and the polymerase chain reaction (PCR) using specific 16S rRNA primers were used to detect and identify the organism and determine its distribution within the soil stratum over the change in seasons. Results showed that Ashdown's method gave higher isolation rates in the dry season, and Galimand and Dodin's method gave higher isolation rates during the wet season. PCR of the soil enrichment proved to be a more sensitive method than culture and was also a useful confirmatory test in determining the identification of isolates where biochemical tests gave inconsistent results. The PCR primers were specific and able to detect 10(1) cfu g-1 soil and 10(4) cfu g-1 of soil using Ashdown's enrichment broth and Galimand and Dodin's broth, respectively. Overall the isolation of B. pseudomallei was greatest during the dry season and at the higher and lower soil depths, which is contradictory to epidemiological evidence that melioidosis occurs primarily during the wet season among patients exposed to contaminated surface soil and water.     

Speed of detection of Burkholderia pseudomallei in blood cultures and its correlation with the clinical outcome

We retrospectively reviewed the cases of 10 consecutive patients treated with hemivertebral excision for congenital scoliosis at The Children's Hospital, Denver, CO, between 1982 and 1992. Follow-up consisted of physical and radiographic examination and averaged 54 months (range, 4-111). Age at surgery ranged from 9 months to 10 years, 5 months (average, 3 years, 11 months). Hemivertebra levels were between T12 and L3. The average preoperative curve measured 40 degrees (range, 20-55 degrees); the average at latest follow-up was 16 degrees (range, 3-37 degrees). We found hemivertebral excision for congenital scoliosis to be a safe and effective means of treatment. Curve correction averaged 67% and seemed to be greatest in those less than 4 years of age at the time of surgery.     

Cellular lipid and fatty acid compositions of Burkholderia pseudomallei strains isolated from human and environment in Viet Nam

Viet nam is known as an endemic area of melioidosis but its etiologic agent originated in Viet nam was not extensively studied. For the first time, we analyzed the cellular lipid and fatty acid compositions of 15 Vietnamese isolates of Burkholderia pseudomallei, 10 from humans and 5 from the environment. Cellular lipid compositions were analyzed by two-dimensional thin-layer chromatography on silica gel G plates. Cellular fatty acid methyl esters were analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The major lipids in all the isolates were phosphatidylglycerol (PG), two forms of phosphatidylethanolamine (PE-1 and PE-2), and two forms of ornithine-containing lipid (OL-1 and OL-2). PE-1 contained non-hydroxy fatty acids at both sn-1 and -2 positions, while PE-2 possessed 2-hydroxy fatty acids and non-hydroxy fatty acids in a ratio of 1:1. Since snake venom phospholipase A2 digestion of PE-2 liberated 2-hydroxy fatty acids, it was confirmed that these acids are at the sn-2 position of glycerol moiety. In both OL-1 and OL-2, amide-linked fatty acid was 3-hydroxy palmitic acid (3-OH-C16:0), while ester-linked fatty acids were non-hydroxy acids in OL-1 and 2-hydroxy acids in OL-2. The total cellular fatty acid compositions of the test strains were characterized by the presence of 2-hydroxy palmitic (2-OH-C16:0), 2-hydroxy hexadecenoic (2-OH-C16:1), 2-hydroxy octadecenoic (2-OH-C18:1), 2-hydroxy methylene octadecanoic (2-OH-C19CPA), 3-hydroxy myristic (3-OH-C14:0) and 3-hydroxy palmitic (3-OH-C16:0) acids. There were significant differences in the concentration of hexadecenoic (C16:1), methylene hexadecanoic (C17CPA), octadecenoic (C18:1) and methylene octadecanoic (C19CPA) acids among the Vietnamese isolates of B. pseudomallei. However, no significant difference was observed in cellular lipid and fatty acid components between strains of human and environmental origins.     

DNA fingerprinting of single colonies of Helicobacter pylori from gastric cancer patients suggests infection with a single predominant strain

In each of six gastric cancer patients, repetitive extragenic palindromic PCR DNA fingerprints of 18 single colonies of Helicobacter pylori from the gastric antrum, corpus, and cardia were identical and matched that of the parental isolate. In three additional gastric cancer patients, 17 of 18 single-colony DNA fingerprints were identical to each other and to the DNA fingerprint of the corresponding parental isolate.     

Clinical and immunological characteristics of transplant recipients with recurrent acute rejection episodes

Diet may play a key role in the pathogenesis of cancer and evidence for the role of a reduced intake of micronutrients with antioxidant properties have been increasingly reported. Until now, epidemiologic research in humans has focused on the negative effect of different diets containing excessive caloric intake and relatively little is known on the possible correlation between slimming diets and the incidence of acute leukemia. In this report we describe the temporal association of imbalanced slimming regimens and the subsequent diagnosis of acute leukemia in three cases. This association may be coincidental or perhaps suggests a possible relationship of the diet with the development or progression of acute leukemia.     

Flagellin gene variation between clinical and environmental isolates of Burkholderia pseudomallei contrasts with the invariance among clinical isolates

The flagellin gene sequence from a clinical isolate of Burkholderia pseudomallei was used to design oligonucleotide primers for PCR/RFLP analysis of flagellin gene variation among clinical and environmental isolates of B. pseudomallei. Genes from four clinical and six environmental isolates were amplified and compared by RFLP. The clinical isolates were indistinguishable, but variation was detected among some of the environmental isolates. Sequence analysis of flagellin gene amplified products demonstrated high levels of conservation amongst the flagellin genes of clinical isolates (>99% similarity), compared to the variation observed between the clinical isolates and one of the environmental isolates (<90% similarity). Genomic comparisons with pulsed-field gel electrophoresis (PFGE) revealed differences between the relationships inferred by flagellin genotyping and PFGE, suggesting that a combination of molecular methods may be useful for the subtyping of B. pseudomallei strains.     

Attachment of Burkholderia pseudomallei to pharyngeal epithelial cells: a highly pathogenic bacteria with low attachment ability

OBJECTIVE: To identify the mutation in a human transforming growth factor beta-induced gene (BIGH3) in a Japanese family with a severe form of granular corneal dystrophy of early onset associated with recurrent corneal erosions. PATIENTS: The tentative clinical diagnosis in this family was Reis-Bücklers corneal dystrophy; 4 persons affected with this disorder have been identified in 4 generations, and 3 of the 4 were examined. The proband underwent keratoplasties in our hospital (Keio University Hospital, Tokyo, Japan). METHODS: The BIGH3 gene was examined for a mutation by the polymerase chain reaction and direct sequencing. Corneal buttons of the proband were stained and examined by electron microscopy. RESULTS: Three affected persons were shown to have a heterozygous G-->T transversion at the second nucleotide position of codon 124 (Arg-->Leu) of the BIGH3 gene. In the proband, corneal deposits between the epithelium and the Bowman layer stained red with Masson trichrome stain. Electron microscopy revealed numerous electron-dense, rod-shaped bodies next to the epithelial basement membrane but no curly fibers suggestive of Thiel-Behnke dystrophy. CONCLUSION: A novel R124L mutation of the BIGH3 gene was associated in this family with a superficial variant of granular corneal dystrophy. CLINICAL RELEVANCE: This mutation causes a severe form of superficial granular corneal dystrophy by producing abnormal keratoepithelin between the epithelium and the Bowman layer and thus clinical similarities to Reis-Bücklers corneal dystrophy.     

Discrimination of Burkholderia gladioli from other Burkholderia species detectable in cystic fibrosis patients by PCR

A procedure for molecular identification of Burkholderia gladioli is described. Specific 16S and 23S rRNA gene signature sequences were defined as primers for PCR. The method allows rapid and specific discrimination of B. gladioli from related species (B. cepacia, B. multivorans, B. vietnamiensis, B. mallei, B. pseudomallei, Ralstonia pickettii, and R. eutropha) and should contribute to the clarification of its role as a human pathogen, e.g., in cystic fibrosis.     

The type II O-antigenic polysaccharide moiety of Burkholderia pseudomallei lipopolysaccharide is required for serum resistance and virulence

Melioidosis, an infection caused by the gram-negative bacterial pathogen Burkholderia pseudomallei, is endemic in south-east Asia and northern Australia. Acute septicaemic melioidosis is a major cause of morbidity and mortality, especially in north-east Thailand. B. pseudomallei is highly resistant to the bactericidal activity of normal human serum (NHS), and we have found that B. pseudomallei 1026b multiplies in 10-30% NHS. We developed a simple screen for the identification of serum-sensitive mutants based on this novel phenotype. Approximately 1200 Tn5-OT182 mutants were screened, and three serum-sensitive mutants were identified. The type II O-antigenic polysaccharide (O-PS) moiety of lipopolysaccharide was not present in the serum-sensitive mutants. A representative serum-sensitive mutant, SRM117, was killed by the alternative pathway of complement and was less virulent than 1026b in three animal models of melioidosis. The Tn5-OT182 integrations in the serum-sensitive mutants were physically linked on the B. pseudomallei chromosome, and further genetic analysis of this locus revealed a cluster of 15 genes required for type II O-PS production. The proteins encoded by these genes were similar to proteins involved in bacterial polysaccharide biosynthesis. The results presented here demonstrate that type II O-PS is essential for B. pseudomallei serum resistance and virulence.     

Determination of genotypes of Toxoplasma gondii strains isolated from patients with toxoplasmosis

To determine the genotypes of Toxoplasma gondii strains associated with human toxoplasmosis, we developed a sensitive approach for typing parasites grown from clinical samples by short-term in vitro culture. A newly described nested PCR assay was capable of amplifying genomic DNA from as few as five parasites in the presence of host tissues. Typing was based on DNA polymorphisms at the SAG2 locus, encoding tachyzoite surface antigen p22. Restriction fragment length polymorphisms in PCR-amplified SAG2 products were used to classify strains into one of the three major lineages of T. gondii. This approach was successfully used to determine the genotypes of 68 of 72 samples that had been previously isolated from patients with congenital, cerebral, and disseminated toxoplasmosis. Type II strains of T. gondii were found in a majority of the samples, accounting for 55 (81%) of the 68 toxoplasmosis cases. In contrast, type I and III strains were found in only 7 (10%) and 6 (9%) of the 68 cases, respectively. The results of this study support the previous finding that type II strains are most often associated with human toxoplasmosis. Nested PCR analysis at the SAG2 locus provides rapid assignment of T. gondii to a specific genotype that should be useful in analyzing a variety of clinical samples.     

The role of anaerobic bacteria in recurrent episodes of sinusitis and tonsillitis

Chronic and recurrent upper respiratory tract infections represent a significant clinical challenge. The causative organisms tend to be heterogeneous, involving both aerobes and gram-positive and gram-negative anaerobes. There is evidence that these mixed groups of bacteria interact synergistically, enhancing and prolonging the overall virulence of infection. The role of anaerobic bacteria, in particular their proposed ability to protect susceptible organisms by the production of beta-lactamases, has been the subject of intense speculation. The evidence of a significant role for anaerobic bacteria in recurrent episodes of tonsillitis and sinusitis is reviewed and the most appropriate antimicrobial strategies and possible future developments in diagnosis and therapy are discussed.     

Identification and characterization of a two-component regulatory system involved in invasion of eukaryotic cells and heavy-metal resistance in Burkholderia pseudomallei

Burkholderia pseudomallei is the causative agent of melioidosis, a disease increasingly recognized as an important cause of morbidity and mortality in many regions of the world. B. pseudomallei is a facultative intracellular pathogen capable of invading eukaryotic cells. We used Tn5-OT182 mutagenesis to generate mutants deficient in the ability to invade a human type II pneumocyte cell line (A549 cells). One of these mutants, AJ1D8, exhibited approximately 10% of the ability of the parental strain, 1026b, to invade A549 cells. There was no difference in the abilities of 1026b and AJ1D8 to resist killing by RAW macrophages or the human defensin HNP-1. The nucleotide sequence flanking the Tn5-OT182 integration in AJ1D8 was determined, and two open reading frames were identified. The predicted proteins shared considerable homology with two-component regulatory systems involved in the regulation of heavy-metal resistance in other organisms. AJ1D8 was 16-fold more sensitive to Cd2+ and twofold more sensitive to Zn2+ than was 1026b but was not sensitive to any of the other heavy metals examined. The B. pseudomallei two-component regulatory system, termed irlRS, complemented the invasion-deficient and heavy-metal-sensitive phenotype of AJ1D8 in trans. There was no significant difference between the virulence of AJ1D8 and that of 1026b in infant diabetic rats and Syrian hamsters, suggesting that the irlRS locus is probably not a virulence determinant in these animal models of acute B. pseudomallei infection.     

Identification and characterization of a novel DNA marker associated with epidemic Burkholderia cepacia strains recovered from patients with cystic fibrosis

Postoperative pain is a common reason for the delayed discharge and unanticipated hospital admission of out-patients. In this study, we examined the pattern of pain in ambulatory surgical patients and determined those factors that predict postoperative pain. Ten thousand eight consecutive ambulatory surgical patients were prospectively studied. Preoperative patient characteristics, intraoperative variables, and pain in the postanesthesia care unit (PACU) and the ambulatory surgical unit (ASU) and 24 h postoperatively were documented. The incidence of severe pain was 5.3% in the PACU, 1.7% in the ASU, and 5.3% 24 h postoperatively. In the PACU, younger male adults (36 +/- 13 vs 47 +/- 22 yr), ASA physical status I patients, and patients with a higher body mass index (26 +/- 5 vs 25 +/- 5 kg) had a higher incidence of severe pain. In the group with severe pain, the duration of anesthesia, the duration of stay in the PACU and the ASU, and the time to discharge was longer than in the group without severe pain. In the PACU, orthopedic patients had the highest incidence of pain (16.1%), followed by urologic (13.4%), general surgery (11.5%), and plastic surgery (10.0%) patients. In patients who had general anesthesia, the intraoperative dose of fentanyl was significantly smaller in the group with severe pain than in the group without severe pain when body mass index and duration of anesthesia were taken into consideration. Body mass index, duration of anesthesia, and certain types of surgery were significant predictors of severe pain in the PACU. This knowledge will allow us to identify those patients at risk of severe postoperative pain and manage them prophylactically. Implications: The pattern of pain was examined in 10,008 consecutive ambulatory surgical patients. The incidence of severe pain was 5.3% in the postanesthesia care unit, 1.7% in the ambulatory surgical unit, and 5.3% 24 h postoperatively. Body mass, duration of anesthesia, and certain types of surgery were significant predictors of pain in the postanesthesia care unit. These data will allow us to better predict those patients who need intense prophylactic analgesic therapy.     

Chronic hypersensitivity lung disease with recurrent episodes of hypersensitivity pneumonitis due to a contaminated central humidifer

A child with a 4-year history of acute and chronic respiratory symptoms of unknown aetiology was investigated for hypersensitivity pneumonitis. Lung disease due to inhalation of material from a contaminated central humidifier was suggested by the clinical history, the presence of precipitating antibodies in the serum against the humidifier water, a pulmonary response to challenge with the humidifier water, and marked improvement after removal of the humidifier. No fungi were cultured from the humidifier nor were antibodies against a number of fungal antigens identified by radioimmunoassay inhibition techniques. Antigenic material was found in the humidifier water and the household water prior to its reaching the humidifier. This antigenic material was not found in laboratory tap water supplied from the same general source (Lake Michigan) but from a different pumping station. Three of the child's siblings gave histories suggestive of a single concurrent episode of acute hypersensitivity pneumonitis and one sibling had a history suggestive of chronic hypersensitivity lung disease. No association could be found between HLA-haplotypy and disease in the patient and the siblings.     

Increase in quinolone resistance in a Haemophilus influenzae strain isolated from a patient with recurrent respiratory infections treated with ofloxacin

The increase in the level of quinolone resistance of Haemophilus influenzae clinical isolates during ofloxacin therapy of a patient with recurrent respiratory infections was investigated. The first isolate (MIC of ciprofloxacin of 2 microg/ml) and the second isolate (MIC of 32 microg/ml) belonged to the same clone, as shown by pulsed-field gel electrophoresis, and the increase in the resistance level was associated with a substitution in Ser-84 to Arg in the ParC protein. These results emphasize the potential risk of development of quinolone-resistant H. influenzae during fluoroquinolone therapy in patients with recurrent respiratory infection.