Genome Structure

Recently, the complete three genomic sequences of the genus Burkholderia have been determined. All are virulent species—B. cepacia, B. pseudomallei, and B. mallei. The B. cepacia (the former name) or Burkholderia cenocepacia genome consisted of three chromosomes of 3.87, 3.22, and 0.88 Mb with a total size of 8.1 Mb. The G+C content of this genome is 66.9%. The genome of B. pseudomallei strain K96243 is 7.24 Mb, with a G+C content of 68.06% consisting of two chromosomes of 4.07 and 3.17 Mb.[12] These genome data have been produced by the B. cenocepacia and B. pseudomallei Sequencing Group at the Sanger Institute and can be obtained from ftp://ftp.sanger.ac.uk/Projects (accessed November 2003). In B. pseudomallei genome, there are approximately 7000 open reading frames, with 60% code for proteins of unknown function. There are no plasmids. The B. mallei strain ATCC23344 has a smaller genome of about 5.9 Mb and 5000 open reading frames.[13] The B. mallei genome data can be obtained from the Institute for Genomic Research (TIGR) Web site at http://www.tigr.org (accessed November 2003). The main differences between B. pseudomallei and B. mallei are in the cell envelope genes, which are more in B. pseudomallei, and in the energy metabolism genes as well. B. pseudomallei also has very few insertion sequences (IS) compared with B. mallei. This is consistent with B. pseudomallei, the ancestral organism of B. mallei, which has become an obligate pathogen.

To date, the proteomic profiles of B. pseudomallei, both extracellular and intracellular proteomes, are under investigation. However, many genes with coding regions have been studied and the sequences are available in the public databases.

IDENTIFICATION AND DIAGNOSIS Bacteriological Examination

Isolation and identification of B. pseudomallei by cultivation remains the method of choice for definitive diagnosis of melioidosis. It is relatively simple and economical to perform. It needs neither elaborate nor expensive equipment but requires experienced personnel, particularly in the interpretation of the results. Its main drawback is that it takes at least 3-4 days to obtain the results, and by that time, it may be too late for successful management, as a high percentage of patients admitted for acute septicemia die within 24-48 hr of admission. Recently, an automatic BacT/Alert® nonradiolabeled blood culture system can be used to overcome this problem. Culture of B. pseudomallei was detected to be positive approximately 62% within 24 hr and 90% within 48 hr.[14] Although the use of a sensitive automated system has considerably cut down the incubation time normally required for the less sensitive semiautomatic or manual systems, its remaining drawbacks are the running cost and its availability only in large hospitals.

Some clinical specimens, such as throat swab and sputum, are generally heavily contaminated by other organisms. The use of selective media, a modified Ashdown medium containing additional chemicals such as colistin on MacConkey's agar or crystal violet, glyc-erol, neutral red, and gentamicin on trypticase soy agar, has been devised for isolation of B. pseudomallei from clinical specimens.[15] Availability of a costly API 20NE test panel has considerably simplified the identification of B. pseudomallei. However, the API test has been reported to misidentify for Chromobacterium violacium and other bacteria.[16]

However, four species—B. pseudomallei, B. mallei, B. cepacia, and B. thailandensis—are similar in many biochemical properties. To differentiate B. pseudomallei from B. mallei, motility assay is the method used to distinguish these organisms because B. mallei is nonmotile as a result of a lack of flagella. To distinguish B. pseudo-mallei from B. cepacia, the Minitek disc system was developed in 1992 as a tool for the differentiation.1-17-1 In case of B. thailandensis, the ability to assimilate L-arabi-nose by B. thailandensis is used to differentiate this organism from B. pseudomallei.[18] At present, other specific methods based on immunology and DNA technology have been developed to use in identification and diagnostic detection of B. pseudomallei.

Immunological Diagnosis

Rapid and specific immunological diagnosis is used to overcome the problem of time-consuming bacteriological examinations. However, regardless of the detection of antibodies or antigens, with the immunological assays, one needs highly specific reagents. Several immunolog-ical assays have been developed and used for the detection of either B. pseudomallei antibodies or antigens (for review, see Ref. [19]).

Antibody detection

The first method is agglutination, which is simple but gives low sensitivity in detecting antibody to B. pseudomallei. In 1970, two methods, the complement fixation test (CF)

and the indirect hemagglutination test (IHA), were developed. An IHA test is still being widely used in many endemic areas of infection. One of the main drawbacks of these two assays that limit their value in clinical situations is the presence of background antibody in some healthy individuals in the endemic area. This is largely attributable to unrecognized subclinical infection in these individuals. In 1981, Ashdown had developed indirect fluorescent antibody staining (IFA) by applying immunofluorescence to detect the antibody in human serum after infection. The IgM antibody could be detected rapidly by fluorescence electroscopy. This method has been very useful for screening of melioidosis. The disadvantages of the method are the expensive microscopy and the need for skilled persons to interpret the results. Enzyme-linked immunosorbent assay (ELISA) has sensitivity, specificity, stable inexpensive reagents, and simple equipment, which is appropriate for use in determination of antibody present in melioidosis patients in developing country. This method has been performed to detect antibody to crude antigen, exotoxin, and endotoxin. Other detection methods such as gold blot have also been developed to detect IgM and IgG antibodies. However, the method has not been evaluated for their clinical usefulness in a large-scale field trial. More recently, recombinant antigens specific for B. pseudomallei have been produced and appeared to be highly specific and useful for the diagnosis of melioidosis.

Antigen detection

This approach is more logical and is superior to antibody detection because it indicates active disease. Many immunological methods have been developed for the detection of B. pseudomallei antigen, including the detection of soluble secreted product in blood and urine, and the detection of whole organisms in pus, wound, sputum, and throat swabs. The first method was undertaken by Ismail et al. in 1987, when they developed a monoclonal antibody-based assay for the quantitation of exotoxin. This assay could detect toxin in the range of 16 ng/mL of culture supernatant fluid. Similarly, in 1990, Wongratanacheewin et al. have developed a polyclonal antibody-based avidin-biotin ELISA which could detect B. pseudomallei antigens in the culture filtrate at a concentration as low as 4 ng/mL. However, both methods have never been evaluated in real clinical situations. More recently, a highly sensitive assay was developed for the detection of antigen, most likely the LPS at a concentration of 12.2 ng/mL, in the urine of patients with systemic and localized infection. When applied to clinical specimens, a sensitivity of 81% and a specificity of 96% have been achieved. The assay is, however, more complicated and more costly to perform and thus may not be very practical for routine use in smaller health centers with a limited health budget. Recently, another antigen-detecting assay based on the use of monoclonal antibody reactive against 200-kDa antigen secreted by B. pseudomallei was developed. This sandwich ELISA detection was shown to be highly reliable giving 75% sensitivity and 95% specificity. Other assay systems have been developed and evaluated including latex agglutination and immuno-fluorescence. The former is not sensitive enough for routine use and needs prior concentration, whereas the latter requires a fluorescent microscope that is not readily available in most laboratories in the endemic area.

Molecular Diagnosis

Because in a medical microbiological laboratory, pathogenic microorganisms particularly need to be identified under killed conditions, molecular diagnosis in different clinical specimens has been successfully used for the diagnosis of a large number of infectious diseases of man and animals and, more recently, evaluated for its reliability in melioidosis. In detection of melioidosis, the hybridization technique does not have sufficient sensitiv-ity[20] and therefore in more recent years, the PCR approach has been more commonly performed with satisfactory results (for review, see Ref. [21]). Different sets of specific primers have been evaluated including the regions in the 23S rRNA, 16S rRNA, a junction between 16S and 23S rRNA, ribosomal protein subunit 21 (rpsU), and specific sequences designed from a specific DNA region. Using these specific primers, as few as a single bacterium present in the clinical specimens such as blood samples[21] or spleen tissue samples[22] can be successfully amplified in laboratory scale and detected with sensitivity approach to 100%. However, none of these has been subjected to critical evaluation in the real clinical situation. Because low numbers of bacteria in samples are suspected to be one of the causes of the low sensitivity of PCR, the amount of bacteria in blood of around half of septicemic melioidosis patients is beyond detection. Adding steps for concentrating the bacteria from blood could definitely improve the sensitivity.[21]

Molecular diagnosis method for discriminating among the three virulent species, B. pseudomallei, B. cepacia, and B. mallei, is more interesting. PCR-RFLP of the flagellin gene sequence has been applied for identification of B. pseudomallei and B. cepacia from the clinical isolates.[23] Surprisingly, the molecular system based on the flagellin gene cannot be applied for differentiation of B. pseudomallei and B. mallei,[24] although B. mallei is a nonmotile species. The PCR-based differentiation of the 23S rDNA sequence between B. pseudomallei and B. mallei has been reported using a species-specific primer in which one nucleotide is substituted for a B. mallei template. Recently, PCR-RFLP based on a specific DNA fragment has been developed to differentiate B. pseudo-mallei and B. mallei.[25] Although these methods have never been evaluated in real clinical situations, these could be applied for epidemiological study and medical microbiological laboratory in identifying under kill conditions or noncultivation pathogenic bacteria as well. More recently, the PCR based on specific flagellin gene primers can be applied to epidemiological identification of B. pseudomallei and B. thailandensis and the mixed population of these two species as well.[26] The difference between the 16S rRNA gene sequences of B. pseudomallei and that of B. thailandensis is only 1%, and it is therefore not discriminative enough to distinguish the two species. The groEL gene sequence is better for distinguishing between B. thailandensis and B. pseudomallei, and the GroEL amino acid and groEL nucleotide sequences of this single gene locus may potentially be useful for a two-tier hierarchical identification of medically important Burkholderia at the genus and species levels.[27]

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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