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Melioidosis in animals: a review on epizootiology, diagnosis and clinical presentation.

Melioidosis, an infectious disease caused by Burkholderia pseudomallei is an emerging disease with high impact on animals and man. In different animal species, the clinical course varies and delayed diagnosis poses risks for the dissemination of the agent in non-endemic areas. Not only migration and transport of animals around the world but also tourism increases the risk that melioidosis can leave its endemic boundaries and establish itself elsewhere. Detection of the agent is a major challenge, as the agent has to be handled in laboratories of biosafety level 3 and test kits are not yet commercially available. Veterinarians and doctors should be aware of melioidosis not only as an agent of public interest but also in terms of a bioterrorist attack. The aim of this review is to describe the agent, its aetiology, the manifestation in a variety of animal species as well as to describe diagnostic procedures, typing techniques and countermeasures.

Animals↗

Cutaneous melioidosis.

Melioidosis is an infection caused by the gram-negative bacterium Burkholderia pseudomallei. This disease is endemic in Southeast Asia and North Australia with sporadic occurrence in temperate countries, mostly imported from travellers. Any organ can be involved in melioidosis whereby Burkholderia pseudomallei causes an acute inflammatory reaction with rapid development of small abcesses which tend to coalesce to form larger abscesses. Cutaneous manifestations vary greatly. We report a man with systemic melioidosis who presented with cutaneous lesions.

Abscess↗

Melioidosis: an imported case from Madagascar.

Burkholderia pseudomallei, the causative agent of melioidosis, is endemic in Southeast Asia and Northern Australia but is a rare pathogen in other parts of the world. No human case of melioidosis has been reported in Madagascar until now. We describe a case of pulmonary melioidosis probably acquired in Madagascar.

Bronchoalveolar Lavage↗

Recombinant truncated flagellin of Burkholderia pseudomallei as a molecular probe for diagnosis of melioidosis.

Current serological tests for melioidosis, using impure or uncharacterized cell antigens from Burkholderia pseudomallei, have problems in detection sensitivity and specificity. Therefore, we designed and expressed the recombinant flagellin (truncated at both the N- and C-terminal ends), and used the antigen to develop an indirect enzyme-linked immunosorbent assay (ELISA) to diagnose melioidosis. Comparison of the immunoreactivities of the full-length and truncated flagellins reveals that the truncated flagellin performed much better in detection specificity and sensitivity. Only the full-length flagellin was recognized by other bacterial causing septicemia and gave a false-positive result in Western analysis, indicating that the cross-reactive epitopes were located on the more highly conserved N- and C-terminal regions of flagellin. The indirect ELISA using recombinant truncated flagellin as the antigen achieved 93.8% sensitivity and 96.3% specificity and offered a more efficient serodiagnosis of melioidosis.

Antigens, Bacterial↗

Melioidosis: epidemiology, pathophysiology, and management.

Melioidosis, caused by the gram-negative saprophyte Burkholderia pseudomallei, is a disease of public health importance in southeast Asia and northern Australia that is associated with high case-fatality rates in animals and humans. It has the potential for epidemic spread to areas where it is not endemic, and sporadic case reports elsewhere in the world suggest that as-yet-unrecognized foci of infection may exist. Environmental determinants of this infection, apart from a close association with rainfall, are yet to be elucidated. The sequencing of the genome of a strain of B. pseudomallei has recently been completed and will help in the further identification of virulence factors. The presence of specific risk factors for infection, such as diabetes, suggests that functional neutrophil defects are important in the pathogenesis of melioidosis; other studies have defined virulence factors (including a type III secretion system) that allow evasion of killing mechanisms by phagocytes. There is a possible role for cell-mediated immunity, but repeated environmental exposure does not elicit protective humoral or cellular immunity. A vaccine is under development, but economic constraints may make vaccination an unrealistic option for many regions of endemicity. Disease manifestations are protean, and no inexpensive, practical, and accurate rapid diagnostic tests are commercially available; diagnosis relies on culture of the organism. Despite the introduction of ceftazidime- and carbapenem-based intravenous treatments, melioidosis is still associated with a significant mortality attributable to severe sepsis and its complications. A long course of oral eradication therapy is required to prevent relapse. Studies exploring the role of preventative measures, earlier clinical identification, and better management of severe sepsis are required to reduce the burden of this disease.

Animals↗

Genome-wide expression analysis of Burkholderia pseudomallei infection in a hamster model of acute melioidosis.

Burkholderia pseudomallei is the causative agent of melioidosis and represents a potential bioterrorism threat. In the current studies we have examined gene expression in B. pseudomallei in an animal model of acute melioidosis using whole-genome microarrays. Gene expression profiles were generated by comparing transcriptional levels of B. pseudomallei-expressed genes in infected hamster organs including liver, lung, and spleen following intraperitoneal and intranasal routes of infection to those from bacteria grown in vitro. Differentially expressed genes were similar in infected livers irrespective of the route of infection. Reduced expression of a number of housekeeping genes suggested a lower bacterial growth rate during infection. Energy production during growth in vivo involved specific biochemical pathways such as isomerization of 3-phosphoglycerate, catabolism of d-glucosamine and inositol, and biosynthesis of particular amino acids. In addition, the induction of genes known to be involved in oxidative phosphorylation including ubiquinol oxidase, ferredoxin oxidoreductase, and formate dehydrogenase enzymes suggested the use of alternative pathways for energy production, while the expression of genes coding for ATP-synthase and NADH-dehydrogenase enzymes was reduced. Our studies have identified differentially expressed genes which include potential virulence genes such as those for a putative phospholipase C and a putative two-component regulatory system, and they have also provided a better understanding of bacterial metabolism in response to the host environment during acute melioidosis.

Animals↗

Clinical evaluation of a type III secretion system real-time PCR assay for diagnosing melioidosis.

A Burkholderia pseudomallei type III secretion system real-time PCR assay was evaluated on clinical specimens in a region where melioidosis is endemic. The PCR was positive in 30/33 (91%) patients with culture-confirmed melioidosis. All six patients with melioidosis septic shock were blood PCR positive, suggesting potential for rapid diagnosis and commencement of appropriate therapy.

Biological Transport↗

Relationships among Pseudomonas pseudomallei isolates from patients with recurrent melioidosis.

Patients with melioidosis may present with recurrent infections after clinical resolution of their primary illness. Because there has been no satisfactory typing scheme for Pseudomonas pseudomallei, recrudescence could not be distinguished from reinfection. We determined the strain identity of primary and relapse isolates of P. pseudomallei from 25 patients with culture-proven melioidosis to answer whether secondary infections were due to the initial infecting strain or to the acquisition of a new strain. Fifty-four isolates were compared by the patterns of BamHI restriction digests produced after hybridization with a cDNA copy of Escherichia coli rRNA. Twenty-three patients had primary and relapse isolates with identical or highly similar ribotype patterns. The patterns of isolates from two patients were different; the primary and relapse isolates differed by a single fragment for one, and the other had identical primary and first-relapse isolates while the second-relapse isolate was markedly different. The results indicated that recurrent infection probably resulted from endogenous relapse in most of the melioidosis patients studied, although reinfection from an exogenous source was also possible in two cases.

Bacterial Typing Techniques↗

Detection of Burkholderia pseudomallei DNA in patients with septicemic melioidosis.

Septicemic melioidosis is the most severe form of melioidosis, which is caused by Burkholderia pseudomallei. It is endemic in Southeast Asia and is the leading cause of death from community-acquired septicemia in northeast Thailand. A major factor that contributes to the high mortality is the delay in isolation and identification of the causative organism. More than half of the patients die within the first 2 days after hospital admission, before bacterial cultures become positive. The present study was undertaken to develop a rapid diagnostic method for identification of this organism. A nested PCR system that amplified a part of 16S rRNA gene that was highly specific to B. pseudomallei was developed. This system was able to detect as few as two bacteria present in the PCR. DNAs from all 30 clinical isolates of B. pseudomallei and none of the other bacteria tested were amplified. The described PCR system has been employed for the detection of the organism in clinical specimens, including buffy coat and pus from internal organs. The detection of B. pseudomallei in buffy coat specimens by PCR was shown to be comparable to the detection of bacteria from blood cultures in septicemic melioidosis cases.

Bacteremia↗

Evaluation of PCR for diagnosis of melioidosis.

Previously published PCR-based diagnostic tests for melioidosis were evaluated for clinical usefulness. A Burkholderia pseudomallei 16S rRNA-derived primer set had a sensitivity approaching 100% for clinical samples from 22 culture-confirmed cases of melioidosis and enabled diagnosis of 3 culture-negative cases. However, samples from 10 of 30 inpatients from Royal Darwin Hospital with other diagnoses were positive by PCR, giving a specificity of 67% and a positive predictive value of only 70%. Although there are a number of intriguing possible explanations for our results, concerns of inappropriate therapy resulting from a positive result by PCR have led us to forgo the advantage of rapid PCR diagnosis for melioidosis until a better system is validated.

Humans↗

Value of throat swab in diagnosis of melioidosis.

Throat swab (TS) cultures were performed for 1,011 patients with melioidosis and 3,524 healthy subjects or patients with other diseases. The specificity of TS culture for the diagnosis of melioidosis was 100%, and the overall sensitivity was 36% (24% for sputum-negative patients and 79% for sputum-positive patients). Direct plating of the TS specimen on Ashdown's medium was rapid (colonies were usually evident within 24 h) but only 63% sensitive compared to the results of primary culture in a selective broth. A throat swab should be cultured in all cases of suspected melioidosis.

Adolescent↗

Evaluation of immunoglobulin M (IgM) and IgG rapid cassette test kits for diagnosis of melioidosis in an area of endemicity.

An enzyme-linked immunosorbent assay-based rapid cassette immunoglobulin G (IgG) and IgM immunochromogenic test kit was compared to the indirect hemagglutination test (IHA) for the diagnosis of acute melioidosis in northeastern Thailand. Admission sera from 70 culture-confirmed septicemic melioidosis patients and 30 patients with localized infections were tested. As a control group, 80 patients with other acute febrile illnesses (other bacterial infections, leptospirosis, or scrub typhus) and 119 healthy individuals were tested. The diagnostic sensitivity of the IgG and IgM tests and the IHA test were 79, 67, and 72%, respectively, with corresponding specificities of 90, 80, and 68%. This kit represents an improvement over IHA for the diagnosis of melioidosis an area of endemicity although, as with other serological tests, it has reduced diagnostic utility in a population with high background seropositivity.

Bacterial Infections↗

Recurrent melioidosis in patients in northeast Thailand is frequently due to reinfection rather than relapse.

Human melioidosis is associated with a high rate of recurrent disease, despite adequate antimicrobial treatment. Here, we define the rate of relapse versus the rate of reinfection in 116 patients with 123 episodes of recurrent melioidosis who were treated at Sappasithiprasong Hospital in Northeast Thailand between 1986 and 2005. Pulsed-field gel electrophoresis was performed on all isolates; isolates from primary and recurrent disease for a given patient different by one or more bands were examined by a sequence-based approach based on multilocus sequence typing. Overall, 92 episodes (75%) of recurrent disease were caused by the same strain (relapse) and 31 episodes (25%) were due to infection with a new strain (reinfection). The interval to recurrence differed between patients with relapse and reinfection; those with relapses had a median time to relapse of 228 days (range, 15 to 3,757 days; interquartile range [IQR], 99.5 to 608 days), while those with reinfection had a median time to reinfection of 823 days (range, 17 to 2,931 days; IQR, 453 to 1,211 days) (P = 0.0001). A total of 64 episodes (52%) occurred within 12 months of the primary infection. Relapse was responsible for 57 of 64 (89%) episodes of recurrent infection within the first year after primary disease, whereas relapse was responsible for 35 of 59 (59%) episodes after 1 year (P < 0.0001). Our data indicate that in this setting of endemicity, reinfection is responsible for one-quarter of recurrent cases. This finding has important implications for the clinical management of melioidosis patients and for antibiotic treatment studies that use recurrent disease as a marker for treatment failure.

Bacterial Typing Techniques↗

Role and significance of quantitative urine cultures in diagnosis of melioidosis.

Melioidosis is associated with significant mortality in countries in which it is endemic. Previous studies have demonstrated that quantitative Burkholderia pseudomallei counts in blood are predictive of mortality. Here we examine the relationship between outcomes and quantitative B. pseudomallei counts in urine. A total of 755 patients presenting to Sappasithiprasong Hospital, Ubon Ratchathani, northeast Thailand (in the northeast part of the country), with melioidosis between July 1993 and October 2003 had quantitative urine cultures performed within 72 h of admission. Urine culture results were divided into the following groups: (i) no growth of B. pseudomallei from a neat sample or pellet, (ii) positive result from a centrifuged pellet only (< 10(3) CFU/ml), (iii) detection of between 10(3) CFU/ml and 10(5) CFU/ml from a neat sample, or (iv) detection of > or = 10(5) CFU/ml from a neat sample. The overall in-hospital mortality rate was 45%. Patients with negative urine cultures had the lowest death rate (39%). Mortality rates rose with increasing B. pseudomallei counts in urine, from 58% for those with positive spun pellets only to 61% for those with between 10(3) CFU/ml and 10(5) CFU/ml and 71% for those with > or = 10(5) CFU/ml. This was independent of age, presence of bacteremia, known risk factors for melioidosis such as diabetes, and the prior administration of antibiotics. The presence of B. pseudomallei in urine during systemic infection is associated with a poor prognosis.

Burkholderia pseudomallei↗

Immunofluorescence microscopy for the rapid diagnosis of melioidosis.

A direct immunofluorescent antibody test (DIF) was developed for the rapid diagnosis of melioidosis, a potentially fatal infection caused by Pseudomonas pseudomallei. In a clinical evaluation of 369 sputum, pus, or urine specimens from 272 patients with suspected melioidosis, the DIF had a sensitivity of 73% and a specificity of 99% compared with culture. Using this DIF, a confident diagnosis of melioidosis can now be made within two hours of admission to hospital, compared with the delay of two to four days required for culture results. Consequent early institution of specific antimicrobial therapy may help to save lives.

Fluorescent Antibody Technique↗

Latex agglutination for rapid detection of Pseudomonas pseudomallei antigen in urine of patients with melioidosis.

A latex agglutination test for the detection of Pseudomonas pseudomallei antigen in urine was evaluated for the rapid diagnosis of melioidosis. With unconcentrated urine, antigen was detected in only 18% of patients with melioidosis overall. However, when urine was concentrated 100-fold, antigen was detected in 47% overall and in 67% of patients with septicaemia or disseminated infection, in whom a rapid diagnosis is most important. The specificity of the test was 100%. These results compared favourably with an enzyme immunoassay. This latex agglutination test is a simple, rapid and highly specific method of diagnosing melioidosis, and will be particularly useful in areas with limited laboratory facilities.

Antigens, Bacterial↗

A comparison of lysis centrifugation, pour plate, and conventional blood culture methods in the diagnosis of septicaemic melioidosis.

AIMS: To determine whether quantitative blood culture methods could improve the diagnosis of septicaemic melioidosis. METHODS: A comparison of conventional broth based blood cultures, a pour plate method, and a commercial lysis centrifugation (Isolator 10) blood culture system was conducted in 71 Thai patients with severe melioidosis. The time to identification of B pseudomallei was recorded for each method. RESULTS: 42 patients (59%) were septicaemic. Compared with conventional blood culture, the Isolator and pour plate methods had sensitivities of 81% and 61%, respectively. The median times to a positive culture were: Isolator 39.3 hours, pour plates 45.5 hours, broth culture 61.8 hours (p < 0.001 Isolator v broth). There was a significant inverse correlation between Isolator tube or pour plate quantitative counts and time to detection (r = -0.44 and -0.57, respectively). Mortality was higher in patients who were septicaemic. CONCLUSIONS: Routine use of one of these quantitative methods, in addition to conventional broth culture, may lead to earlier diagnosis of septicaemic melioidosis.

Adolescent↗

Reactivation of latent melioidosis.

Reports of melioidosis in residents of European countries are rare. We describe a case of reactivation of latent melioidosis in a United Kingdom resident. The case demonstrates the lack of clinical response to chemotherapy despite proven in vitro sensitivity of the organism to the drugs used. It is important to consider melioidosis as a cause of septicaemic illness in patients who have travelled to, or been resident in South-East Asia.

Anti-Bacterial Agents↗