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1.
FEMS Microbiol Lett ; 271(2): 170-9, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17403048

ABSTRACT

Molecular typing of leptospiral strains based on variation within putative O-antigen polymerase gene (wzy) was determined among reference strains and those isolated from patients. Using the PCR primers designed from the flanking gene of wzy derived from Leptospira interrogans serovar Copenhageni, all L. interrogans serovars as well as human and rodent leptospiral isolates from Thailand could be amplified. The size of PCR product ranged from 1 to 1.5 kb. The limitation of these primer pairs was the inability to amplify those strains whose sequences differ in the region of the primers, these included Leptospira biflexa (serovar Patoc), Leptospira borgpetersenii (serovar Tarassovi) and Leptospira kirschneri (serovar Bim, Bulgarica, Butembo). Notably, amplification was not limited to L. interrogans as demonstrated by the amplification of some strains from L. kirschneri, Leptospira meyeri, Leptospira noguchii, Leptospira santarosai, L. borgpetersenii and Leptospira weilii. The phylogenetic tree of wzy sequence, inferred by posterior probability of the Bayesian, enabled the categorization of leptospiral serovars into seven genetically related group, of which its differentiation power was better than that of the more highly conserved 16S rRNA gene, which is used extensively for genotyping.


Subject(s)
Hexosyltransferases/genetics , Leptospira/genetics , RNA, Ribosomal, 16S/genetics , Animals , Genotype , Humans , Leptospira/classification , Leptospira/isolation & purification , Leptospirosis/microbiology , Molecular Sequence Data , Phylogeny , Sequence Analysis, DNA , Thailand
2.
Antimicrob Agents Chemother ; 51(3): 1038-42, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17194824

ABSTRACT

In a randomized controlled trial of amphotericin B-based therapy for human immunodeficiency virus (HIV)-associated cryptococcal meningitis in Thailand, we also compared the mycological efficacy, toxicity, and pharmacokinetics of oral versus intravenous flucytosine at 100 mg/kg of body weight/day for the initial 2 weeks. Half of 32 patients assigned to the two arms containing flucytosine were randomized to oral and half to intravenous flucytosine. Early fungicidal activity was determined from serial quantitative cultures of cerebrospinal fluid (CSF), and toxicity was assessed by clinical and laboratory monitoring. Flucytosine and fluorouracil concentrations in plasma and CSF were measured by high-performance liquid chromatography. No significant bone marrow or hepatotoxicity was seen, there was no detectable difference in bone marrow toxicity between patients on intravenous and those on oral formulation, and no patients discontinued treatment. In patients receiving intravenous flucytosine, the median 24-h area under the concentration-time curve was significantly higher than in the oral group. Despite this difference, there was no difference in early fungicidal activity between patients on intravenous compared with patients on oral flucytosine. The results suggest that either formulation can be used safely at this dosage in a developing country setting, without drug concentration monitoring. The bioavailability of the oral formulation may be reduced in late-stage HIV-infected patients in Thailand. Concentrations of flucytosine with intravenous formulation at 100 mg/kg/day may be in excess of those required for maximal fungicidal activity.


Subject(s)
Antifungal Agents/administration & dosage , Antifungal Agents/therapeutic use , Flucytosine/administration & dosage , Flucytosine/therapeutic use , HIV Infections/complications , Meningitis, Cryptococcal/drug therapy , Administration, Oral , Adult , Antifungal Agents/pharmacokinetics , Area Under Curve , Female , Flucytosine/pharmacokinetics , Hemoglobins/metabolism , Humans , Injections, Intravenous , Male , Meningitis, Cryptococcal/etiology , Meningitis, Cryptococcal/microbiology , Microbial Sensitivity Tests
3.
J Immunol ; 174(3): 1746-50, 2005 Feb 01.
Article in English | MEDLINE | ID: mdl-15661940

ABSTRACT

In animal models, immunity to cryptococcal infection, as in many chronic fungal and bacterial infections, is associated with a granulomatous inflammatory response, intact cell-mediated immunity, and a Th1 pattern of cytokine release. To examine the correlates of human immunity to cryptococcal infection in vivo, we analyzed immune parameters at the site of infection over time and assessed the rate of clearance of infection by serial quantitative cerebrospinal fluid (CSF) fungal cultures in 62 patients in a trial of antifungal therapy for HIV-associated cryptococcal meningitis. CSF IL-6, IFN-gamma, TNF-alpha, and IL-8 were significantly higher in survivors compared with nonsurvivors. There were negative correlations between log TNF-alpha, IFN-gamma, and IL-6 levels and baseline cryptococcal CFU. Log IFN-gamma, G-CSF, TNF-alpha, and IL-6 were correlated positively with the rate of fall in log CFU/ml CSF/day. In a linear regression model including antifungal treatment group, baseline CFU, and these cytokines, only treatment group and log IFN-gamma remained independently associated with rate of clearance of infection. The results provide direct in vivo evidence for the importance of quantitative differences in IFN-gamma secretion in human immune control of granulomatous infections, and increase the rationale for adjunctive IFN-gamma in the treatment of refractory HIV-associated cryptococcosis.


Subject(s)
Interferon-gamma/metabolism , Meningitis, Cryptococcal/immunology , Cryptococcus neoformans/growth & development , Cryptococcus neoformans/immunology , Granulocyte Colony-Stimulating Factor/biosynthesis , Granulocyte Colony-Stimulating Factor/cerebrospinal fluid , HIV Infections/cerebrospinal fluid , HIV Infections/drug therapy , HIV Infections/immunology , HIV Infections/mortality , Humans , Inflammation Mediators/cerebrospinal fluid , Inflammation Mediators/metabolism , Interferon-gamma/cerebrospinal fluid , Interleukin-10/biosynthesis , Interleukin-10/cerebrospinal fluid , Interleukin-6/biosynthesis , Interleukin-6/cerebrospinal fluid , Interleukin-8/biosynthesis , Interleukin-8/cerebrospinal fluid , Meningitis, Cryptococcal/cerebrospinal fluid , Meningitis, Cryptococcal/drug therapy , Meningitis, Cryptococcal/mortality , Multivariate Analysis , Prognosis , Time Factors , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/cerebrospinal fluid
4.
Mol Cell Probes ; 16(3): 217-22, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12144773

ABSTRACT

We have previously shown that Burkholderia pseudomallei, the causative pathogen of melioidosis, may be discriminated from the closely related non-pathogenic species Burkholderia thailandensis by the presence of a 15 base pair deletion in the flagellin gene of B. thailandensis. Using specific flagellin gene primers flanking the distinctive region, PCR products of 191 and 176 bp in size were detected for B. pseudomallei and B. thailandensis, respectively. The sensitivity of detection is 20-80 colony forming units/reaction of B. pseudomallei and B. thailandensis cell suspension. To mimic the expected environmental situation, mixed populations of the two species were analyzed. The results showed that the PCR-based method could be use to distinguish the two species in a duplex reaction. In addition, we have developed a simplified method for direct PCR-based detection from soil samples. The result indicated that about 200 colonies of bacteria per reaction could be detected. This method can be applied to epidemiological studies, especially for investigating the ecological relationship between these two species in the environment.


Subject(s)
Burkholderia/classification , DNA Primers/standards , DNA, Bacterial/analysis , Flagellin/genetics , Asia, Southeastern , Burkholderia/genetics , Burkholderia Infections/diagnosis , Burkholderia pseudomallei/genetics , Humans , Polymerase Chain Reaction , Sensitivity and Specificity , Soil Microbiology/standards
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