Immune responses of selected phagotopes from monoclonal antibodies of Burkholderia pseudomallei.

Random peptide libraries displayed by bacteriophage T7 and M13 were employed to identify mimotopes from 4 monoclonal antibodies (MAbs) specific to Burkholderia pseudomallei. Insert DNA sequences of bound phages selected from four rounds of panning with each MAb revealed peptide sequences corresponding to B. pseudomallei K96243 hypothetical protein BPSL2046, hypothetical protein BpseP_02000035, B. pseudomallei K96243 hypothetical protein BPSS0784, B. pseudomallei 1710b hypothetical protein BURPS1710b_1104, and B. cenocepacia H12424 TonB-dependent siderophore receptor, all located at the outer membrane. The immune responses from all selected phagotopes were significantly higher than that of lipopolysaccharide. The study demonstrates the feasibility of identifying mimotopes through screening of phage-displayed random peptide libraries with B. pseudomallei MAbs.

Multiple mutations in katG and inhA identified in Thai isoniazid-resistant Mycobacterium tuberculosis isolates.

A total of 29 Thai multi-drug-resistant/isoniazid-resistant Mycobacterium tuberculosis isolates were analyzed for mutations in katG from codons 254 to 549, inhA promoter and inhA open reading frame by DNA sequencing and single strand conformation polymorphism. Twenty-five multi-drug resistant isolates exhibited single point mutations (17 isolates at Ser315Thr plus Arg463Leu, 1 at Thr308Pro plus Arg463Leu, 7 at either Ser315Thr or Arg463Leu) while the other 4 isoniazid-resistant isolates had single point mutation only at Arg463Leu. Seven of 25 multi-drug-resistant isolates [4 at C(-15)T, 1 at T(-8)C; 1 at C(-15)T plus Ser94Ala and 1 at Ile21Val] and 2 of 4 isoniazid-resistant isolates [1 at C(-15)T, 1 at C (-15)T plus Ile21Thr] had mutations in inhA promoter and open reading frame, while the other 20 isolates had no mutation at any position. No frame shift mutation was observed in any tested isolates. This is the first report of two mutations, Trp308Pro of katG and T (-8)C of inhA in Mycobacterium tuberculosis isolates.

Mutation in the rpoB gene of the rifampicin resistant M. avium complex strains from Thailand.

Forms of mutation never before described in the rpoB gene are reported for a sample of 20 rifampicin-resistant Mycobacterium avium Complex (MAC) strains isolated from AIDS patients in Thailand. All strains were analyzed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and polymerase chain reaction-DNA sequencing (PCR-DNA sequencing). Sequence analysis of these strains revealed that only one strain (5%) has missense mutation at Lys-626 (Thr) and the rest of the strains had 15 different silent mutations within a 542 bp region of the rpoB gene. Five strains (25%) had a silent mutation at only one position, 7 (35%) at 2 positions, 7 (35%) at 3 positions, and 1 (5%) at 7 positions. The silent mutation at the Ala-630 codon occurred in the largest proportion of the strains (15 strains, 75%), followed by the Val-581 in 8 strains (40%), Tyr-578 and Thr-600 in 4 strains (20%), and Gly-597 in 3 strains (15%). This investigation demonstrates that mutation in the rpoB gene of MAC strains from Thailand are more varied than previously reported for RIF MAC strains. PCR-SSCP screening clearly separated RIFr strains from rifampicin-susceptible (RIFs) strains.

Development of a combined air sampling and quantitative real-time PCR method for detection of Legionella spp.

The objective of this study was to develop and optimize the combined methods of air sampling and real time polymerase chain reaction (real-time PCR) for quantifying aerosol Legionella spp. Primers and TaqMan hydrolysis probe based on 5S rRNA gene specific for Legionella spp were used to amplify a specific DNA product of 84 bp. The impinger air sampler plus T-100 sampling pump was used to collect aerosol Legionella and as low as 10 fg of Legionella DNA per reaction could detected. Preliminary studies demonstrated that the developed method could detect aerosol Legionella spp 1.5-185 organisms /500 l of air within 5 hours, in contrast to culture method, that required a minimum of 7-10 days.

Preparation of in vivo cow control blood samples for cadmium analysis.

Quality control is essential for any analysis in the laboratory. The objective of this study was to prepare in vivo cow control blood samples. The experiment was performed by feeding cows with a single dose of cadmium in the form of cadmium chloride, withdrawing the blood at an appropriate time to get the highest level of cadmium and detecting the level of cadmium in the blood. It was found that feeding the cow a single dose of 0.06 mg cadmium per kg body weight resulted in the highest cadmium level of 3.622 microg/l 30-60 minutes after feeding. The samples were homogeneous because feeding the cows with single dose of cadmium let the cadmium be absorbed and distributed naturally. In addition, the samples were stable during transport. Therefore, they may be used as quality control samples to detect cadmium levels without using a lyophilized process. They could be used for proficiency testing and to evaluate whole blood analysis in the laboratory.

Mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains from Thailand and its evolutionary implication.

Novel mutations in the rpoB gene are reported for 70 rifampicin-resistant (RIFr) M. tuberculosis strains from Thailand. Sequence analysis of these strains revealed mutations in a 435 base-pair region of the rpoB gene. Twenty-eight strains (40%) had single mutations, and 26 of those strains had mutations at positions never before reported, of which, just one had a substitution at Val-432 (Asp), and the remaining 25, a silent mutation at Gln-517. All other strains had multiple mutations, of which 24 (34%) had mutations at two positions; 9(13%), at three positions; 2(3%), at five positions; and 1(1%) at six positions. Five strains (7%), reported to have the RIFr phenotype, contained no mutation in the examined region of the rpoB gene. Surprisingly, one RIFr strain had silent mutations at 29 positions. By far the dominant mutation was the silent mutations at Gln-517 (86%). This investigation demonstrates that mutations in the rpoB gene of M. tuberculosis strains from Thailand are more varied than previously reported for RIFr M. tuberculosis strains. Screening by means of PCR-SSCP clearly separated RIFr strains from rifampicin-susceptible (RIFs) strains. There was no correlation between RIFr mutations and random amplified polymorphic DNA (RAPD) types.

Mutations in the gyrA and gyrB genes of fluoroquinolone-resistant Mycobacterium tuberculosis from TB patients in Thailand.

Among fluoroquinolone-resistant Mycobacterium tuberculosis (FQr-MTB) isolates, mutation at positions 90, 91, and 94 in gyrA gene and at positions 495, 516, and 533 in gyrB gene have been frequently reported. In this study, 35 isolates of FQr-MTB were collected from Siriraj Hospital and Chest Disease Institute. The quinolone-resistance-determining regions (QRDR) of gyrA and gyrB genes in all 35 FQr-MTB isolates and from the H37Ra MTB strain were amplified using polymerase chain reaction (PCR). DNA-sequencing and single-strand conformation polymorphism (SSCP) were further utilized for characterization of the mutations in the QRDR of gyrA and gyrB genes and mutation screening, respectively. From DNA-sequencing, 21 of 35 (60%) exhibited single-point mutations in different positions, at Ala90Val, Ser91Pro, and Asp94(Gly/Ala/His/Asn); and one novel mutation position at Gly88Cys in the gyrA gene and Asp495Asn in the gyrB gene. These positions were previously frequently reported to be responsible for FQr-MTB. The other 14 FQr-MTB isolates (40%) had no mutation. This study is the first report of mutation occurring only in the QRDR of the gyrB gene, without prior mutation in the gyrA QRDR among FQr-MTB isolates. By SSCP analysis for screening of the mutant FQr-MTB, the SSCP patterns of mutated FQr-MTB isolates were clearly differentiated from the SSCP patterns of FQs-MTB.

Epitope mapping of monoclonal antibodies specific to serovar of Leptospira, using phage display technique.

Random heptapeptide library displayed by bacteriophage T7 was used to characterize epitopes of five monoclonal antibodies that were specific to L. australis, L. bangkok, and L. bratislava. Phages selected by biopanning were cloned by plaque isolation, and the binding specificity of individual clones was confirmed by enzyme-linked immunosorbent assay, before being further amplified and checked for phage peptide sequence using PCR and DNA sequencing. Almost all of the peptide epitopes were continuous or linear. Interestingly, in phages reacting with the monoclonal antibody (MAb) clones F11, F20, 2C3D4, and 8C6C4A12, the deduced amino acid sequence of the displayed peptides corresponded to a segment of hypothetical protein of the Leptospira genome (L. interrogans serovar Lai and Copenhageni). Considering the deduced amino acid sequences of phages reacting with the MAb clones F11, F20, 2C3D4, and 8C6C4A12, the consensus motif -SKSSRC-, -TLINIF-, -SSKSYR- and -CTPKKSGRC- appeared respectively. No similarity was observed among phage reacting with the MAb clone F21. The results demonstrate that T7 phage display technique has potential for epitope mapping of leptospiral MAbs, and for rapid analysis of the interactions between phage display peptides with the MAb. The finding of a phage peptide that binds to MAb with protective activity can be further tested as a candidate for leptospirosis vaccine in the future.

Comparative studies on the pathological findings and mortality in Schistosoma mansoni infected mice after treatment with artesunate and the current antischistosomal drugs.

The effect of artesunate (ART) on the pathology and mortality rate of in Schistosoma mansoni infected mice was comparatively studied with the current drugs of choice for the treatment of schistosomiasis mansoni: praziquantel (PZQ) and oxamniquine (OX). S. mansoni experimentally infected mice were treated at 9th week of infection with ART, PZQ or OX at an oral dosage of 300 mg kg(-1), 600 mg kg(-1) and 100 mg kg(-1), respectively. Untreated, infected mice and non-infected mice were added as controls. Samples of mice were sacrificed and examined for the pathological findings at 1 week, 1 month, and 3 months after treatment. At 1 week after treatment, both gross and microscopic lesions were observed. No significant differences were noted among the infected groups. Differences were observed at 1 month after treatment. The lesions decreased more rapidly in groups treated with PZQ and OX. At 3 months after treatment, there were significant differences in the pathological findings among groups. In the groups treated with PZQ and OX, the lesions were markedly reduced and rarely found, but they were clearly observed in the group treated with ART and in the untreated, infected group. High mortality was also recorded in the group treated with ART and in the untreated, infected group. Therefore, the treatment of S. mansoni infected mice at 9 weeks of infection with ART did not reduce the pathological findings or the mortality rate compared to treatment with the current recommended schistosomicides, PZQ and OX.

Therapeutic effect of subcurative dose praziquantel on Schistosoma mansoni infected mice and resistance to challenge infection after treatment.

The therapeutic effect of a subcurative dosage of praziquantel (PZQ) on Schistosoma mansoni infected mice and resistance to challenged worm infection after treatment were assessed and compared with conventional treatment using a curative dosage of PZQ. S. mansoni infected mice were treated with PZQ at a curative dosage (600 mg kg(-1)) or a subcurative dosage (300 mg kg(-1)) at 9 weeks after infection. Untreated mice and non-infected mice were added as controls. The therapeutic effect of the drug was evaluated in terms of the mortality of mice after treatment, and the parasitological and pathological findings in mice sacrificed at 1 week, 1 month, or 3 months after treatment. Another sample of mice was not killed but challenged with S. mansoni cercariae at 1 week, 1 month, or 3 months after treatment. Resistance to re-infection was evaluated by the extent of challenged worm reduction. In conclusion, there was no significant difference in mortality, or parasitological and pathological findings between mice treated with PZQ at the two dosages. However, resistance to challenged worm infection was more sustained in the group treated with subcurative dose PZQ, especially at 3 months after treatment.