The optimization and development of an ELISA-disc procedure for the detection of antibodies to whole cell surface antigens and purified exotoxin ofPseudomonas pseudomallei is described. Comparison of the serum agglutination test (SAT), the serum based enzyme-linked immunosorbent assay (ELISA) and the ELISA-disc procedures used on goat and human sera demonstrated a high correlation in their ability to detect antibodies specific forP. pseudomallei antigens. A serological survey using the ELISA-disc method was carried out on a normal human population in Sabah, Malaysia, an area known to be endemic for melioidosis. The prevalances of antibodies towards cell surface antigens and exotoxin ofP. pseudomallei were 28% and 8%, respectively. As a procedure, the ELISA-disc technique reported here is technically simple and provides savings in costs and is thus deemed suitable for seroepidemiological surveillance of melioidosis in remote areas of South-East Asia.
We report a rare case of suppurative epididymo-orchitis caused by Pseudomonas pseudomallei in a 56-year-old male. This is a gram negative bacillus found mainly in tropical zones. Diagnosis was reached by culture of the organisms after drainage of the scrotal abscess, and the patient was treated by a course of oral chloramphenicol 500 mg qid for 6 months.
The occurrence of latency and relapse in human melioidosis suggests adaptations by Burkholderia pseudomallei that help to avoid the human immune response. Ruthenium red-stained preparations of bacterial cultures viewed by electron microscopy revealed three morphologically distinct variants; one with a very marked and another with a less electron-dense layer surrounding the cell wall, and a third variety devoid of such a structure. This structure may be attributable to a layer of polysaccharide, suggesting the presence of a glycocalyx that may aid in the survival of the organism during latency.
In order to assess the usefulness of immunohistochemistry in the diagnosis of melioidosis, an infection by Burkholderia pseudomallei, polyclonal antibodies were applied to tissues from known cases of melioidosis and to other infected tissues. Formalin-fixed, paraffin-embedded tissues were stained by a modified immunoperoxidase technique. In autopsy tissues with inflammatory lesions of melioidosis, the cytoplasm of phagocytes and intact bacilli, both intra- and extracellular, were stained very strongly positive. Relatively more focal positive staining was observed in some but not all surgical biopsies from proven cases of melioidosis. In granulomas staining was mainly found in the central necrotic areas, with little staining of peripheral phagocytes. All control materials stained negative. Immunohistochemistry appears to be a useful diagnostic tool in melioidosis.
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.
Forty-nine isolates of Burkholderia pseudomallei from sporadic cases of melioidosis in Malaysia over the past 18 years were examined by BamHI ribotyping and pulsed-field gel electrophoresis (PFGE) of XbaI digests of total deoxyribonucleic acid (DNA). Twenty-four patients had septicaemic melioidosis with a mortality of 70%; mortality in the non-septicaemic disease was 16%. Five ribotype patterns were identified, 2 of which accounted for 90% of all isolates. PFGE revealed a number of different strains within these ribotypes, but some pairs of isolates from unrelated cases gave closely similar DNA profiles. These results are in agreement with Australian studies which showed a high prevalence of a few ribotypes of B. pseudomallei which are further divisible by genotyping, in areas where melioidosis is endemic.
Melioidosis is endemic in Malaysia. Emerging resistance with new and current antimicrobial agents has underscored the need to look further for new antimicrobial agents for the treatment of melioidosis. Hence, we evaluated the in-vitro susceptibility of fifty locally isolated strains of Burkholderia pseudomallei, the causative agent of melioidosis to cefoperazone-sulbactam combination using the method of NCCLS. All the fifty strains tested were susceptible in-vitro to cefoperazone-sulbactam. The MIC90 of the organism for cefoperazone-sulbactam was 4 mg/L. The results of our findings suggested that cefoperazone-sulbactam may be useful in the treatment of melioidosis.
We report a case of meningitis and one of fatal septicaemia in neonates due to Burkholderia pseudomallei and review the literature on neonatal melioidosis. Pneumonia was the primary presentation and was complicated by shock in the latter case. The epidemiological findings suggest that the cases reported from Malaysia were community-acquired in contrast with those from the USA and Thailand.
The constancy of strain genotypes of multiple isolates of Burkholderia pseudomallei from 13 patients with melioidosis was examined by BamHI ribotyping and pulsed-field gel electrophoresis (PFGE) of XbaI digests of DNA. Seven of 8 patients with single episodes of melioidosis each yielded genetically identical isolates and only one of five patients with recurrent episodes was infected with a new strain clearly distinct from the original primary strain. Variation was observed in PFGE patterns of primary and relapse isolates of another patient but this was insufficient to define genetically distinct strains. We conclude that most patients with single or multiple episodes of melioidosis retain a single strain.
Melioidosis is a protean disease caused by Burkholderia pseudomallei. It is rare in the UK and is generally only seen in patients with a travel history to endemic areas such as Thailand, Singapore and Malaysia. Cases may present with disseminated bacteraemic, non-disseminated bacteraemic, multi-focal bacteraemic or localized disease. Subclinical infections also occur. Following acquisition of the organism a patient may remain asymptomatic for several years before infection becomes clinically apparent. Factors such as diabetes, renal failure or other causes for a decrease in host immunity may precipitate the appearance of overt disease. The current treatment choice for severe melioidosis is parenteral ceftazidime followed by oral amoxycillin-clavulanic acid or a combination of co-trimoxazole, doxycycline and chloramphenicol. We report a case of melioidosis in a 59-year-old male diabetic from Bangladesh who initially responded to piperacillin-tazobactam but was changed to ceftazidime when a definitive diagnosis was made. His condition deteriorated on the latter antibiotic. He subsequently responded to imipenem. The patient's long-term outcome is still not known.
We describe a convenient, versatile and safe method for preparing bacterial DNA for ribotyping analysis. In this method, extraction of bacterial DNA from Salmnonella typhi and Burkholderia pseudomallei. and subsequent restriction endonuclease digestion, was performed in agarose blocks/plugs thus minimizing shearing and loss of DNA, problems commonly associated with liquid phase phenol extraction. Digested DNA in the plugs was then electrophoresed directly, transferred to nylon membranes and hybridized with labeled rDNA probes in the usual manner to provide reproducible restriction patterns. This method is particularly useful for bacterial species where standard DNA extraction in the liquid phase using phenol has been problematic (e.g. B. pseudomallei) but can be used for any bacterial species. The DNA extracted within the agarose plugs can be stored for long periods and can be used in other, widely-used typing methods such as pulsed-field gel electrophoresis (PFGE) and PCR-based techniques. Embedding live cells directly in agarose plugs also minimizes the risk of exposure to these virulent human pathogens among laboratory workers.
Seven isolates of Burkholderia pseudomallei from cases of melioidosis in human (2 isolates) and animal (2 isolates), cat (one isolate) and from soil samples (2 isolates) were examined for in vitro sensitivity to 14 antimicrobial agents and for presence of plasmid DNA. Randomly amplified polymorphic DNA (RAPD) analysis was used to type the isolates, using two arbitrary primers. All isolates were sensitive to chloramphenicol, kanamycin, carbenicillin, rifampicin, enrofloxacin, tetracycline and sulfamethoxazole-trimethoprim. No plasmid was detected in all the isolates tested. RADP fingerprinting demonstrated genomic relationship between isolates, which provides an effective method to study the epidemiology of the isolates examined.
Melioidosis caused by Burkholderia pseudomallei is endemic in southeast Asia. The clinical manifestations range from wound infections to acute septicemia. In some cases, recurrence can also occur following complete recovery. Case fatality rates are high and a major factor is the delay in the culture and identification of the bacterium. An immunofluorescent assay (IFAT) using whole-cell antigen for the detection of total antibodies to B. pseudomallei was tested with 650 sera. Using a cut-off value of 1:80, 66 sera from culture-confirmed cases were positive with titers > or = 320. In another 523 sera from patients in which no other etiology could be found, 149 (23.4%) were positive. To monitor disease activity, persistence of antibody levels was investigated on 61 serial sera samples collected from 14 other confirmed cases on follow-up visits while on oral maintenance therapy. The IFAT demonstrated a reduction in titers in cases of localized infections, suggesting that either the infection was being resolved or arrested while septicemic patients maintained high IFAT titers on follow-up, suggesting the possibility of continuous sequestration of antigen from an intracellular source.
There is remarkably little known about the incidence of melioidosis outside a few countries (Thailand, Australia, Singapore and Malaysia). Presumably it is widespread in tropical south east Asia. Elsewhere there are tantalising glimpses of the tip of what may be a large iceberg. Since a specific diagnosis of melioidosis requires awareness on the part of clinicians, and the existence of a laboratory capable of isolating and identifying Burkholderia pseudomallei, a luxury not available in most rural tropical areas, the size of this iceberg is likely to remain unknown for the foreseeable future. There is mounting evidence that the disease is endemic in the Indian sub-continent and the Caribbean, and there have been unsubstantiated reports of recent cases in South Africa and the Middle East. It is unclear whether melioidosis has really spread to such areas relatively recently, or has been there but unrecognised for a long time. Almost all cases diagnosed in temperate climates have been imported from the tropics, with the exception of a unique outbreak which occurred in France in the mid-1970s. With increasing world wide travel of both humans and other animals, the potential exists for melioidosis to spread to new and fertile pastures.
Four species of synanthropic flies were trapped in downtown Kuala Lumpur: Chrysomya megacephala, Chrysomya rufifacies, Musca domestica, and Musca sorbens. Burkholderia pseudomallei, the organism causing melioidosis, was the dominant bacteria isolated from Chrysomya megacephala. Klebsiella oxytoca, commonly associated with nosocomial infections, was commonly isolated from Chrysomya megacephala, Musca domestica, and Musca sorbens. Aeromonas hydrophila, the bacteria causing gastroenteritis, was predominantly isolated from Chrysomya megacephala and also from Musca domestica and Musca sorbens. A total of 18 bacterial species was isolated from the synanthropic flies trapped. Burkholderia pseudomallei had been reported for the first time.
A total of 35 Burkholderia pseudomallei isolates from Thailand (16 clinical and eight soil isolates) and Malaysia (seven animal, two isolate each from clinical and soil) were investigated by their antimicrobial resistance, plasmid profiles and were typed by randomly amplified polymorphic DNA analysis. All isolates were found to be resistant to six or more of the 12 antimicrobial agents tested. Only two small plasmids of 1.8 and 2.4 megadalton were detected in two clinical isolates from Thailand. RAPD analysis with primer GEN2-60-09 resulted in the identification of 35 RAPD-types among the 35 isolates. The constructed dendrogram differentiated the 35 isolates into two main clusters and a single isolate. The wide genetic biodiversity among the 35 isolates indicate that RAPD-PCR can be a useful method to differentiate unrelated B. pseudomallei in epidemiological investigation.
IgM and IgG based ELISA systems were developed using the culture filtrate antigen (CFA) of Burkholderia pseudomallei. The assays were evaluated using 95 sera from 66 septicemic cases and 47 sera from 20 cases with localized melioidosis. In addition 65 sera from culture negative cases that were also serologically negative for other endemic infections clinically suspected of melioidosis were included. These were compared with sera from 260 non-melioidosis cases, 169 sera from individuals with high risk of acquiring the infection and 48 sera from healthy controls. The IgG-ELISA was 96% sensitive and 94% specific. All sera from cases with septicemic and localized infections and 61 of 63 sera from clinically suspected melioidosis cases were positive for IgG antibody. The geometric mean titre index (GMTI) values of IgG antibody in melioidosis cases were significantly higher (p < 0.0005) compared to that of healthy subjects, high risk group and subjects with non-melioidosis infections. The sensitivity and specificity of IgM ELISA was 74 and 99% respectively. The GMTI value of IgM antibody in the sera of melioidosis cases was significantly higher as compared to that of non-melioidosis disease controls (p < or = 0.001). These results demonstrate that the detection of IgG is a better indicator of the disease in the diagnosis of melioidosis.
The class and subclass distribution of antibody response to the culture filtrate antigen (CFA) of Burkholderia pseudomallei was examined in the sera of 45 septicaemic and 17 localised melioidosis cases and 40 cases clinically suspected of melioidosis and the results were compared with those from high-risk and healthy control groups. The geometric mean titre index (GMTI) values for all classes and subclasses of immunoglobulins examined were higher for sera from the proven and clinically suspected melioidosis cases than for the control groups. However, the highest response in the three patient groups was that of IgG with GMTIs ranging from 219.4 to 291.6 and the lowest was for IgM with GMTIs of 22.5, 24.3 and 28.7. The IgA response was intermediate with GMTIs ranging from 119.2 to 170. The GMTIs were highest for IgG in septicaemic and localised infections and for IgA and IgM in localised infections. As regards IgG subclass distribution, IgG1 and IgG2 were the predominant subclasses produced against the CFA in contrast to IgG3 and IgG4, which were produced in low amounts. None of the sera from the control groups had any significant titres of antibodies.
In melioidosis caused by Burkholderia pseudomallei, although every organ in the body may be involved, the highest mortality of 73% occurs when the respiratory system is affected. These patients invariably die of acute respiratory failure. Most of them also have underlying predisposing factors like diabetes mellitus.
Melioidosis is a potentially deadly infection that can affect any organ system. Reports of melioidosis of the ENT/head and neck region are relatively uncommon. Four cases are presented: (i) parotid abscess evolving into necrotising fasciitis, (ii) acute sinusitis and parapharyngeal cellulitis resulting in upper airway obstruction, (iii) acute suppurative lymphadenitis (iv) and chronic suppurative otitis media causing meningoencephalitis. Three of the four cases are believed to be unique, as a literature review of melioidosis in ENT/head and neck is also presented. Some practical issues of management are also discussed. Not suspecting melioidosis does not change contemporary empirical broadspectrum antibiotic therapy. The value of suspicion or on confirmation of diagnosis lies in anticipating and planning for rapid change.