phenotypic identification of burkholderia pseudomallei

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The crucial need and importance to make a correct diagnosis of melioidosis has always been a problematic dilemma for both the clinicians and laboratory scientist. To date, this still relies on isolation and accurate identification of the pathogen from clinical specimens. Culture is still the gold standard even though a delay of 24 to 48h or more between the time of specimen plating and bacterial growth plus presumptive identification often occurs. Phenotypic identification of Burkholderia pseudomallei is often delayed since laboratory personnel still unfamiliar with the bacterium and may overlook it as Pseudomonas sp. (Leelarasmi and Bovornkitti, 1989). Several other serologic and molecular methods have been tested, but not clinically evaluated. Despite a plethora of research and scientific papers, using many different diagnostic tests in order to get the most reliable result is considered not realistic in the clinical applications.

In this present study, we successfully cultured 35 isolates (32 clinical, and three environmental) through out the period of study. The isolation rate was higher with the use of Francis medium. Viability of the organism during transport of the specimens was not a problem since almost all of them were from within HTAA, and some nearby district hospitals. Moreover, they were transported to our research laboratory in its transport medium. Initially, we cultured the samples and evaluated the characteristics of the isolates on three different media, blood agar, Macconkey agar, and Francis agar. Growth on blood agar revealed that most of the clinical strains but all environmental strains were beta-hemolytic. Observation on MacConkey media showed reddish colonies. This probably because of oxalic acid production from an amino acid rather than fermentation of lactose as described in Gilligan et. Al (1999).

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On Francis medium, B. pseudomallei grew rapidly producing colonies with a yellow haze along the primary streak lines after 18 hours of incubation. This rapid growth rate differentiates the organism from B. mallei, which typically takes a minimum of 72 hours to grow. This medium was were proven to act both a selective and differential one while Macconkey and blood agar act as selective medium only. In clinical specimen like pus, which is generally heavily contaminated by other organisms, B. pseudomallei can be overgrown by contaminating flora, as the generation time for B. pseudomallei is considerably longer than that of other common bacteria. Thus, selective inhibition of the organism is essential for the recovery of B. pseudomallei.

Our results are in accord with these of Francis et al (1999) which confirmed that the use of Francis medium enables the hospital microbiology laboratory to give an early preliminary diagnosis of melioidosis within 24 hrs of receiving a clinical specimen. We also found that the medium is easy to prepare from easily obtainable materials at low cost. However, some training is needed to read the plates. Even in spite of this improvement in culture technique, most studies showed that culture dependent diagnostic procedures give delayed results that are obtained too late to influence clinical management (Chaowagul et. al,1993). Thus, confirmation from certain biochemical tests are still needed to confirmed the diagnosis and this will approximately lead to 2 to 3 days of specimens turn-around time. Conventional biochemical tests and API20E substrate-utilization test panel [bioMιrieux] kit used for identification of B. pseudomallei; however, can easily misidentify  Chromobacterium violaceum (Inglis et. al., 1998). This is where the vital part of our research suits in.

In this present project, we were able to diagnose melioidosis by culture, characterize the isolates and compared it with diagnosis done by IFAT and PCR. Phenotypic characterization of this isolates was done according to the criteria described for gram negative non-fermentative bacilli (Ashdown 1979). All isolates were shown to be motile with bipolar staining characteristics. The bipolar description was probably because of intracellular deposits of B-hydroxy butyric acid (Inglis et al. 2001, Sprague & Neubauer 2004). All strains formed thick pellicles seen at the broth-air interface. This is possibly because of positive aerotaxis of B. pseudomallei, and reflects a type of a multicellular organization that resembles a biofilm (Viriginio et al, 2006). All isolates showed typical biochemistry reactions for B. pseudomallei but vary for assimilation of certain glucose.

B. pseudomallei and a closely related species Burkholderia thailandensis, can be distinguished by their ability to assimilate the aldopentose, L-arabinose. The L-arabinose-non assimilators such as B. pseudomallei, are highly virulent and can be isolated from both clinical specimens and the environment. The L-arabinose assimilators, now known as B. thailandensis, however, are usually avirulent and found mainly in the environment (Brett et al. 1998). B. pseudomallei isolates were tested for arabinose assimilation by growth on minimal salts agar containing 0.2% L-arabinose. None of the clinical isolates of B. pseudomallei could assimilate arabinose. This was demonstrated by absence of growth on minimal salts medium in 48 h. Although Ara+ B. pseudomallei has been reported to cause infection (Lertpatanasuwan et al. 1999), all isolates causing melioidosis in our study were Ara-. Our findings are in accord with previous observations described by Vuddhakul et al. (1999) in Thailand and by Miralles et al. (2004) in Brazil.

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Various techniques have been applied as complimentary diagnostic tools beside culture. A frequent employed test is serology, looking for specific antibodies. Serology is useful to monitor disease activities and relapse. Early serological tests developed by Nigg in 1963 were the complement fixation test and the haemagglutination test (IHAT) using crude extracts of B. pseudomallei as antigen. Strauss et. al., 1969 did a seroprevalence study by the IHAT using an antigen from cultured supernantant termed melioidin. Their findings indicated wide spread occurrence of antibody to B. pseudomallei in the human population living in endemic areas (ranging from 2-16%). A study in north-eastern Thailand has shown the indirect haemagglutination test (IHA) to have a sensitivity of 95% but a specificity of only 59% by using a cut-off level of 1:20 dilution. In that study, some of the non-melioidosis septicaemia patients had positive titres of more than 1:1,280. This high antibody titre may persist for a long time after infection subsides. However, acute seroconversion in clinically septic patient from endemic areas strongly suggests melioidosis.

Latex agglutination test was claimed to be a useful approach, being simple, rapid, and applicable both to isolates and broth cultures. Latex agglutination with a monoclonal antibody specific for the extracellular polysaccharide of B. pseudomallei has the advantage of avoiding cross-reactivity with B. thailandensis (Steinmetz et al., 1999; Wuthiekanun et al., 2002). However, since the latter species is never isolated from clinical cases of melioidosis [the very rare arabinose-assimilating clinical isolates reported not having been fully characterized (Lertpatanasuwan et al., 1999)], this is really only a problem when dealing with environmental isolates. Unfortunately, these reagents are not commercially available.

A form of indirect fluorescent antibody technique (IFAT) was initiated by Ashdown, 1981 to develop an IgG and IgM immunofoluerescent test for melioidosis. Thus, the second phase in our study was to revise and evaluate the IFAT serodiagnostic tool. The technique involves formation of an antigen-antibody complex detected by fluorescein-conjugated anti-human immunoglobulin antibody. In our study, the sensitivity and specificity of IFAT were 93.75% and 75%, respectively. These values were slightly lower than previous study by Naigowit et al., 1992. The lower values here may be due to the effect of earlier empirical antibiotic treatment or due to presentation for medical care earlier in the course of the illness, both of which result in lower bacterial loads and consequently lower antibody response. Efforts to increase sensitivity would be improved by centrifuging the specimen and staining the pellet, but this would likely impact on the non-specific background and cost, and, therefore, needs to be re-assessed in another study. Thus, false negatives did occur in our study, and some centres also have not completely resolved this problem. The negative results for some patients in the present study are presumably due to the delayed collection of sera. This is apparent in the cases of two patients, one recovering from acute septicemic melioidosis and the other from chronic localized melioidosis but late in admission to the hospital. Though immunoflurorescent antibody assay is said to be rapid, highly sensitive and specific test for the identification of current infection, it requires a fluorescent microscope that is not always available in some laboratories in endemic areas.

Regardless of many deficiencies, most study showed that IFAT was more useful than the IHAT in differentiating between positive and negative reactions (Naigowit et al, 1992). This finding was confirmed by Vadivelu et al, 2000 who showed that the IFAT was more sensitive and specific than IHAT especially amongst various high risk groups such as diabetics, pyrexics, pregnant women as well as farmers.

Another study done by Vadivelu et al, 1995, suggested that the IFAT was practical in monitoring the progression of the disease during maintenance therapy since patients with localized non-septicemic infections and on maintenance therapy demonstrated progressive reduction in titers. Moreover, these findings also demonstrate the need to follow-up patients with melioidosis, even though they are clinically asymptotic, until there is serologic evidence that the infection has resolved. In addition in a healthy immunocompetent, young individual given appropriate therapy at an early stage, it may be possible to overcome the long-term survival of the bacterium in vivo.

The specificity of the IFAT in our study was 75% which was lower compared to almost 99% done by Wuthiekanun, 2005 where but they used slightly modified IFAT techniques. The small number of false-positive results may reflect the presence of nonviable organisms affected by prior antibiotic administration. In the case of our study, five healthy blood donors were detected positive by IFAT. This may be due to exposure of the persons to this infection before. Another problem with serology was that the background of positive serology in the general population restricted its usefulness in an endemic area. The level of titre that is considered as diagnostic will show a discrepancy in different locations according to local melioidosis epidemiology. Cross-reactions with infections caused by other Burkholderia and Pseudomonas species, as well as with Legionella species have been reported by Kunakorn, Boonma, Khupulsup, and Petchclai, 1990.

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Moreover, we did agree that IFAT is relatively labor intensive and takes more than 2h to complete. In small clinical laboratory or centre, the use of IFAT was mostly limited since it required the fluorescent microscope to read the results. Thus, the use of IFAT was not practical in clinical health setting especially in rural areas.

Theoretically, immunofluorescence technique generally employs two sets of antibodies: a primary antibody is used against the antigen of interest; a subsequent, secondary, dye-coupled antibody is introduced that recognizes the primary antibody. In this presence study, the IFAT method applied involved the use of whole-cell antigen for the detection of total antibodies to B. pseudomallei, using a cut-off value of 1:80. In some cases, the researcher may create several primary antibodies that recognize various antigens, but, because they all share a common constant region, may be recognized by a single dye-coupled antibody. Typically this is done by using antibodies made in different species. For example, a researcher might create antibodies in a goat that recognize several antigens, and then employ dye-coupled rabbit antibodies that recognize the goat antibody constant region (denoted rabbit anti-goat). This allows re-use of the difficult-to-make dye-coupled.

Moreover, the use of primary antibodies directly labelled with a fluorophore is an advantageous. This direct labelling decreases the number of steps in the staining protocols. More importantly, it often avoids cross-reactivity and high background problems. Nowadays, fluorescent labelling can be performed in less than one hour with readily available labelling kits.

As with most fluorescence techniques, a significant problem usually occurred in immunofluorescence is photobleaching. Loss of activity caused by photobleaching can be minimized by reducing the intensity or time-span of light exposure, and by increasing the concentration of fluorophores. In addition, it is advisable to use more robust fluorophores that are less prone to bleaching. Further study need to be done to modify and improved this valuable technique to shorten diagnostic time-length and increase the sensitivity. Despite some pros and contra in the method, the IFAT method is still very useful for prognostic purposes and maintenance therapy.

In the attempts to make an early diagnosis without relying on cultures, several attention have centered on methods to detect the specific antibodies or antigen of this organisms. This crucial needs lead to the development of an alternative method for early detection which is amplification of specific DNA sequences by the polymerase chain reaction, PCR. The term 'chain reaction' refers to several cycles of copying a specified stretch of DNA from a target nucleic acid, in this case from the genome of an infectious agent. This method requires short, specific fragments of DNA (oligonucleotides) to act as primers. PCR primers are design either from published DNA sequence or genome database when available or from related species when some degeneracy may be allowed in the oligonucleotide. The use of DNA polymerase, a heat stable enzyme, which does not denature during heat cycling make it possible to copy the DNA sequence between the primers. Nucleic acid amplification is performed in a thermocycler, which is an instrument that can hold the assay's reagents and allows the reactions to occur at the various temperatures required.

In the initial step of the procedure, nucleic acid (e.g., DNA) is extracted from the microorganism or clinical specimen of interest. Heat (90C-95C) is used to separate the extracted double-stranded DNA into single strands (denaturation). Cooling to 55C then allows primers specifically designed to flank the target nucleic acid sequence to adhere to the target DNA (annealing). Following this, the enzyme Taq polymerase and nucleotides are added to create new DNA fragments complementary to the target DNA (extension). This completes one cycle of PCR. This process of denaturation, annealing and extension is repeated numerous times in the thermocycler. At the end of each cycle each newly synthesized DNA sequence acts as a new target for the next cycle, so that after 20-40 cycles millions of copies of the original target DNA are created. The result is the accumulation of a specific PCR product with sequences located between the 2 flanking primers. By repeating 20-40 times a heat-cycling regime, the amount of copied target DNA gained is enough for further operations, such as detection, cloning or sequencing.

Comprehensive researches have been done on PCR to implement it as diagnostic tool for melioidosis, but the efficient results on clinical application is still insufficient. Thus, we evaluated two PCR based system in this project. The first method involved extraction of bacterial DNA from the culture colonies before PCR amplifications. The second method involved another PCR method to detect B. pseudomallei directly from the clinical specimens. For this, we use the primers named 'LPS' (Rattanathongkom et al., 1997) which were selected from regions in specific DNA clones obtained from the result of cross hybridization with other bacterial DNA (Sermswarm et al, 19994) for clinical applications. The sensitivity and specificity of this method were calculated as compared to the diagnostic gold standard, Culture.

For the first PCR system, we effectively managed to detect B. pseudomallei in 32 cases out of 35 positive cultured cases. Twenty other closely related organisms were tested as negative controls. Our results showed sensitivity of 93.75 % and specificity of approximately 100%. The performance of this test has been comparable to the report of previous study, sensitivities and specificities of 95%-98% and 98%-100% respectively (Lew & Desmarchelier, 1994). Our lower sensitivity of PCR technique were presumably due to the smaller conserved regions of primers targeted to B. pseudomallei compared to the previous study and purifications of DNA techniques need further improvement in our laboratory. The high specificity in this system was probably contributed from specific targets of the primers and lower risk of carry-over products and contaminations applied. Stringent precautions have been adopted in our laboratory although there is always a risk for contamination in PCR assays.

The second PCR system was directly done from clinical specimens such as blood and pus. This PCR system was evaluate for clinical setting with the expectations to overcome the prolonged diagnosis duration by culture. DNA extractions were done from the EDTA-blood tubes of the patients. For this, the salting method was applied to increase the DNA yield. The control group included twenty blood samples from septicemia patients other than B. pseudomallei. In this presence study, the sensitivity and specificity were 90.6%and 95% respectively.

The sensitivity of this PCR system was considered high as compared to other direct-from-specimen PCR study. Kunakorn et al, 2000 reported sensitivity and specificity of 31.03% % and 98% respectively. Our results were also comparable with study done by Meumann, Ryan, Novak, Mirjam, Kaestli, Mayo, Hanson, Emma Spencer, Glass and Gee (2006) resulted sensitivity and specificity of 90.6% and 100% respectively.

Regarding specificity, there were concordant false positive results considering the mixed infection of B. pseudomallei with other bacteria is possible in the endemic areas of melioidosis. In mixed infections, the presence of presence present were relatively low compared with other bacteria (Werner et al, 1967) that grow faster, result in overgrowth of other bacteria, with the presence of B. pseudomallei being missed in the culture. It is therefore suggested not to exclude the presence of B. pseudomallei in some other patients in the control group that may be responsible for the 'false positive' results in PCR in this study group.

One culture-negative but PCR-positive sample was obtained from melioidosis patients. This probably usually represents the detection of nonviable bacteria, as the majority of these culture-negative but PCR-positive samples were taken following commencement of specific melioidosis therapy. Meanwhile, the false-positive results correspond from non-melioidosis patients is still ambiguous but possibilities include contamination, cross-reactivity with an alternative DNA sequence, detection of undiagnosed melioidosis, or asymptomatic B. pseudomallei carriage.

Moreover, this PCR assay can be completed within 1 day, whereas cultures for B. pseudomallei typically require up to 5 days for identification of the bacteria. In addition to these diagnostic applications, nucleic acid amplification procedures can also be modified to allow for the quantitative measurement of bacterial load in order to monitor response to antibiotic therapy.

Although there is always a risk for contamination in PCR assays, stringent precautions taken in our laboratory is likely to explain the high sensitivity. These included autoclaving all the apparatus needed, one way workflow in the laboratory, dedicated biologic safety cabinets and the use of aerosol-resistant pipette tips. It is very important to use aerosol-resistant pipette tips; otherwise, false positive results are almost always the rule (even trace amounts of these targets provide a sufficient number of copies to allow amplification to work). All the pipetting tasks were done under a laminar flow of sterile air.

We have also included some optimization of the PCR to improve its sensitivity. Other potentially beneficial factors in this study included repurification of the DNA after extraction, optimizing Mg2+ concentration, annealing temperature and extension time. Theoretically, it is useful to keep the vials on ice while pipetting the ingredients of the reaction to minimize the chance of primer binding to the DNA template and to prevent the polymerase from working (even) prior to the first denaturing step. The nucleotides were frozen in aliquots, thaw quickly and keep on ice once thawed. This is to avoid multiple freeze-thaw cycles. In addition, we always keep in mind to perform a positive control reaction with a template/primer combination that has amplified well in the past to determine when one reaction component was omitted. It is cardinal to ensure the thermal cycler was programmed suitably by verifying that times and temperatures. Small changes in cycling conditions can affect the yield of products.

Thus, our report managed to exemplify the usefulness of PCR-mediated typing of B. pseudomallei from a clinical perspective. We currently recommend using this LPS PCR system not only for blood, but to be tested with sputum, urine, cerebrospinal fluid in another research. In summary, the LPS PCR assay directly done from clinical samples showed sensitive and specific detection of B. pseudomallei. It is practical since the assay has the potential to make a rapid diagnosis in patients with melioidosis septic shock.

Additional evaluation is required in other locations where melioidosis is endemic, with testing of samples from multiple tissue sites in parallel with culture. Their effects will be significant in acute-care settings where timely and accurate diagnostic tools are critical for patient treatment decisions and outcomes.

To date, PCR is the most well-developed molecular technique and has a wide range of already fulfilled, and potential, clinical applications. This enabled microbiologists to define disease by the presence of virulence, toxin, or antimicrobial resistance genes and to identify potentially important clones of organisms responsible for outbreaks of infection (Louie M, Read, Simor, Louie L, 2000). PCR-based methods may also be cost effective relative to traditional testing procedures. Additional advancement of technology is needed to improve automation, optimize detection sensitivity and specificity. Another advance method in identifying infectious disease which is multiplex-PCR is said to increase the capacity to identify multiple targets simultaneously (GrÃ-ndahl, Puppe, Hoppe, KÜhne, Weigl and Schmitt, 1999).

These tools have been developed in response to diagnostic methods that lack sensitivity, specificity, or rapid turnaround time, to assist with identification of agents that are difficult to cultivate or classify or as methods for assessing the effects of antiviral or antimicrobial agents in chronic infection. Similar types of gene identification can be useful to verify or detect genes responsible for phenotypic characteristics, whereas modified forms of the PCR enable whole genome searches for genetic polymorphisms among strains of a given species and sub-species-level DNA fingerprinting (Fredricks and Relman, 1999). In medical sciences, both strategies, gene and genome variability analysis by PCR, have an increasing impact on the study of the spread of especially those microbes that are multiply resistant to clinically used antibiotics.

Conclusions

Overall, our study proved that the difference in phenotypic characterizations didn't represent virulency. Both D- arabinose positive and D-arabinose negative strains were characterized in clinical isolates of the patients. The use of Kirby- Bauer disc diffusion test for detecting B. pseudomallei antibiotic profiles may be practical as a limited screening tool in poor settings laboratory, since no exact standard of interpreataion has been established for this organism. Further evaluation by MIC methodology is advisable.

Thus, our report managed to represent the effectiveness and clinical values of PCR-mediated typing of B. pseudomallei from medical settings. We suggested this LPS PCR system not only for blood, but to be tested with sputum, urine, cerebrospinal fluid in another research to compliment the culture method. In summary, the LPS PCR assay directly done from clinical samples showed higher sensitive and specific detection of B. pseudomallei. Moreover, this assay took about five to six hours to complete and running this test towards many samples simultaneously will decrease the cost of labor. The assay has the potential to make a rapid diagnosis in patients with melioidosis septic shock. When applied selectively in the laboratory, these applications can enhance diagnostic approaches and clinical management and will probably evolve into standard laboratory and point-of-care testing protocols.

Further study need to do typing of the resistance patterns of this organism. Advance studies are needed to determine the degree of bacteremia when detecting B. pseudomallei in the blood. Their effects will be significant in acute-care settings where timely and accurate diagnostic tools are critical for patient treatment decisions and outcomes.