Rheumatological manifestations in patients with melioidosis.

Melioidosis, an infection caused by the bacterium Burkholderia pseudomallei, has a wide range of clinical manifestations. Here, we describe rheumatological melioidosis (involving one or more of joint, bone or muscle), and compare features and outcome with patients without rheumatological involvement. A retrospective study of patients with culture-confirmed melioidosis admitted to Sappasithiprasong Hospital, Ubon Ratchathani during 2002 and 2005 identified 679 patients with melioidosis, of whom 98 (14.4%) had rheumatological melioidosis involving joint (n=52), bone (n = 5), or muscle (n = 12), or a combination of these (n=29). Females were over-represented in the rheumatological group, and diabetes and thalassemia were independent risk factors for rheumatological involvement (OR; 2.49 and 9.56, respectively). Patients with rheumatological involvement had a more chronic course, as reflected by a longer fever clearance time (13 vs 7 days, p = 0.06) and hospitalization (22 vs 14 days, p < 0.001), but lower mortality (28% vs 44%, p = 0.005). Patients with signs and symptoms of septic arthritis for longer than 2 weeks were more likely to have extensive infection of adjacent bone and muscle, particularly in diabetic patients. Surgical intervention was associated with a survival benefit, bur not a shortening of the course of infection.

Polymorphism in the promoter region of tumor necrosis factor-alpha gene is associated with severe meliodosis.

Melioidosis is an important infectious disease endemic in Southeast Asia and the Northern territories of Australia. Septicemic melioidosis, is the leading cause of fatality from community acquired septicemia in northeastern part of Thailand where death often occurs within a few days after hospitalization. The present study was carried out to investigate the polymorphisms of the position -308 promoter region of the TNF-alpha gene, as well as of the intron 1 of the TNF-beta gene in patients with melioidosis compared with normal uninfected controls in the same endemic area. The gene frequency of TNF2 allele was significantly higher in melioidosis patients compared with control subjects (p = 0.0097, relative risk 2.32). The increase in TNF2 allele in melioidosis patients was found in both heterozygous and homozygous forms. In addition, the increase in TNF2 allele was most apparent in patients who had fatal outcome from septicemic melioidosis (p = 0.017), but was also observed with lesser degree in other groups of melioidosis patients. However, no difference in the frequency of TNF-beta polymorphism the melioidosis patients was observed.

HLA-DR and -DQ associations with melioidosis.

Melioidosis is an important infectious disease of southeast Asia caused by an intracellular bacterium, Burkholderia pseudomallei. Cellular immunity is postulated to play important roles in immunity to melioidosis that may influence the severity and clinical outcome of the disease. The present study was undertaken to investigate possible associations of melioidosis with HLA class II alleles. HLA typing of HLA-DRB1, -DQA1, and -DQB1 was performed using polymerase chain reaction and sequence-specific oligonucleotide hybridization (PCR-SSO). Seventy-nine melioidosis patients and 105 healthy, ethnically and geographically matched controls were studied. Among 24 DRB1 alleles, 7 DQA1 alleles, and 13 DQB1 alleles identified in this population, an association with melioidosis was observed with DRB1*1602 which was increased in melioidosis patients (10.1%) compared to normal controls (4.8%), p = 0.047 (odds ratio (OR) = 2.25). In addition, significant increase of DRB1*1602 allele frequency and decrease of DQA1*03 were also observed in septicemic melioidosis patients, the most severe form of the disease (p = 0.01, OR = 3.10; and p = 0.047, respectively). Furthermore, a trend of association of DRB1*0701, DQA1*0201, and DQB1*0201 with relapse cases of melioidosis was also noted. In contrast, no HLA association was observed in localized melioidosis or melioidosis with diabetes mellitus. These findings provide the suggestive evidence of an immunogenetic basis of certain aspects of melioidosis.

The epidemiology of melioidosis in Ubon Ratchatani, northeast Thailand.

BACKGROUND: Melioidosis, or infection with Pseudomonas pseudomallei is an important cause of morbidity and mortality in South East Asia and Northern Australia. The epidemiology of melioidosis in Ubon Ratchatani, Northeast Thailand was studied over a 5-year period from 1987 to 1991. METHODS: Rates and, when possible, the risks of developing melioidosis were calculated. The numerator was the number of culture-proven cases of melioidosis seen in the 1000-bed referral hospital of the province. The denominators were obtained from the population census, a survey of Health, Welfare and Use of Traditional Medicine, and the North Eastern Meterological Centre, Thailand. RESULTS: The average incidence of human melioidosis was 4.4 (95% confidence interval [CI]: 3.8-5.0) per 100,000. The disease affected all ages with the highest incidence in 40-60 years olds. Melioidosis was 1.4 (95% CI: 0.4-5.3) times more common in males than females. The disease showed a significant seasonal variation in incidence, and a strong linear correlation with rainfall (r = 0.7, 95% CI: 0.5-0.9) Adults exposed to soil and water in their work (most were rice farmers) had an increased risk of melioidosis (in the 40-59 year age group, relative risk = 4.1, 95% CI: 2.4-6.9). Most adult patients had an underlying disease (mainly diabetes mellitus) predisposing them to this infection. CONCLUSION: Melioidosis may result from either acute exposure to the organism in the soil and water, or 're-activation' of an asymptomatic childhood infection (by an unidentified possibly infective seasonal cofactor). The results from this analysis are consistent with both hypotheses. Further epidemiological studies are needed to identify risk factors so that optimal strategies for control of melioidosis may be developed.

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.

Maintenance therapy of melioidosis with ciprofloxacin plus azithromycin compared with cotrimoxazole plus doxycycline.

This is a report of a randomized, open, labeled study of the maintenance treatment of melioidosis using a combination of ciprofloxacin and azithromycin (Regimen A) for 12 weeks versus a combination of cotrimoxazole and doxycycline (Regimen B) for 20 weeks. The study was conducted at two tertiary-care hospitals in northeast Thailand. A total 65 patients were enrolled, 36 and 29, respectively, between August 1997 and July 1998. Subjects were randomly allocated to each arm of the trial, resulting in 32 treated under Regimen A and 33 in B. The main outcome was a culture-proven relapse in melioidosis. There were more relapses under Regimen A at 22% (7 of 32) than in Regimen B, 3% (1 of 33). The 19% difference in the rates was significant (95% confidence interval [CI]: 3% to 34%; exact P-value = 0.027). Based on our data, a combination of cotrimoxazole and doxycycline treatment for 20 weeks should be given further consideration as the maintenance therapy of choice for melioidosis.

Diagnostic value of an antibody enzyme-linked immunosorbent assay using affinity-purified antigen in an area endemic for melioidosis.

Melioidosis, an infection caused by Burkholderia pseudomallei, is endemic in southeast Asia. The septicemic form of melioidosis is the leading cause of death from nonhospital-acquired septicemia in the northeastern part of Thailand. A major factor that contributes to the high mortality is the delay in isolation and identification of the causative organism. The present study was undertaken to evaluate the use of enzyme-linked immunosorbent assays based on an immunoaffinity-purified antigen for detecting specific IgG and IgM antibodies to this organism as a rapid serodiagnostic method for melioidosis. The diagnostic value of these tests was evaluated in an actual clinical situation in an area endemic for melioidosis. The specificity of specific IgG test (82.5%) and the specific IgM test (81.8%) were significantly better than that of the indirect hemagglutination (IHA) test (74.7%). The sensitivity of the specific IgG assay (85.7%) was higher than that of the IHA test (71.0%) and the specific IgM test (63.5%). Specific IgG antibody was detected in a majority of septicemic melioidosis (87.8%), as well as in localized forms (82.6%). The specific IgG test was also better than the specific IgM test and the IHA test in identifying acute melioidosis cases in the first five days after admission. In addition, the IgG antibody level to this antigen remained high over a period of more than five years in those who had recovered from melioidosis and remained clinically free of the disease. These results indicate that the detection of specific IgG antibody is clinically useful for the diagnosis of acute melioidosis in an endemic area.

The use of selective media for the isolation of Pseudomonas pseudomallei in clinical practice.

Ashdown's selective-differential agar medium, with or without preenrichment in selective broth, was evaluated for the isolation of Pseudomonas pseudomallei from 1972 clinical specimens obtained from 643 subjects in Northeast Thailand; 226 patients proved to have meliodosis. The use of Ashdown's medium significantly increased the frequency of recovery of P. pseudomallei from sites or specimens with an extensive normal flora (throat, rectum, wounds and sputum) as compared to the recovery on blood and MacConkey agars (p less than 0.01). The isolation frequency from throat, rectal and wound swabs was further increased by the use of the broth pre-enrichment. The colonial morphology of P. pseudomallei on Ashdown's medium was sufficiently characteristic to allow presumptive identification. With the use of these selective media it was possible to culture P. pseudomallei from throat swabs taken from 87% of the patients from whom the organism could also be isolated from corresponding tracheal aspirates or sputum specimens. P. pseudomallei was isolated from rectal swabs taken from 51 patients, the first time that faecal excretion of the organism has been demonstrated in man. The diagnosis of melioidosis would not have been confirmed bacteriologically in eight patients (3.5%) without the use of the selective media. It is suggested that, in areas endemic for melioidosis, all sputum specimens should be cultured on selective media, such as Ashdown's. For the investigation of clinically suspected cases of melioidosis, and for follow-up during treatment of the disease, the use of broth pre-enrichment is recommended for specimens obtained from sites with an extensive normal flora.

Oral fluoroquinolones for maintenance treatment of melioidosis.

Ciprofloxacin (20 mg/kg/d) or ofloxacin (12 mg/kg/d) given for a median of 15 weeks (range 12-40) were used for maintenance treatment of 57 adult patients with melioidosis. The median duration of follow-up in the 45 patients who complied with treatment and were followed for at least 6 months was 28 months (range 6-65). Fluoroquinolone treatment was well tolerated. There were 13 treatment failures (5 failures to respond, 8 relapses), a failure rate of 29% (95% confidence interval 17-43%). The median time to treatment failure was 7 months (range 2-26). These results are inferior to those with courses lasting 20 weeks of amoxycillin/clavulanic acid or the combination of chloramphenicol, doxycycline and trimethoprim/sulphamethoxazole, and suggest that the fluoroquinolones should be reserved as third line agents, and not used for the maintenance treatment of melioidosis unless there is resistance to, or intolerance of, the other available antimicrobial compounds.

Amoxycillin-clavulanic acid treatment of melioidosis.

Melioidosis is a serious infection with high acute mortality, and a high rate of relapse despite protracted antimicrobial treatment. The current recommended conventional oral treatment regimen is a 4-drug combination of high-dose chloramphenicol, doxycycline and trimethoprim-sulphamethoxazole given for between 6 weeks and 6 months. We have evaluated prospectively the use of amoxycillin-clavulanic acid, to which Pseudomonas pseudomallei is consistently sensitive in vitro, for the oral maintenance treatment of melioidosis. Amoxycillin-clavulanic acid was used either as sole treatment of localized disease, or as maintenance therapy following either parenteral ceftazidime or the conventional 4-drug regime; 20 patients with localized infections and 26 with septicaemic melioidosis received a median of 7.5 (2-12) weeks treatment. After a mean follow-up period of 6 months (range 1-19), 31 patients (67%) remain free of disease. The drug was well tolerated. Three patients had fatal relapses, one other died suddenly at home, and another died from underlying promyelocytic leukaemia. The remaining 10 relapses were treated successfully. Resistance developed in one case. Amoxycillin-clavulanic acid is a safe alternative to the conventional 4-drug antimicrobial combination for the oral treatment of melioidosis. It may be of particular value in children, pregnant women, and in infections with Ps. pseudomallei resistant to the potentially toxic conventional regimen, but the optimum dose and duration of therapy need to be established.

The use of bone marrow culture for the diagnosis of melioidosis.

We have evaluated prospectively the contribution of bone marrow culture to the diagnosis of melioidosis. Bone marrow (BMC) and blood cultures (BC) were collected concurrently from 105 patients with suspected acute, severe melioidosis. 67 patients were subsequently proved to have the disease whilst other significant organisms were isolated from these specimens in 5 cases. Overall, 67.2% of BC and 64.2% of BMC from melioidosis patients grew Pseudomonas pseudomallei. Time to positivity did not differ significantly in paired BC and BMC specimens. These results do not support the routine use of BMC in the diagnosis of acute, severe melioidosis. In one patient with pulmonary melioidosis, however, blood cultures were repeatedly negative, whilst bone marrow grew P. pseudomallei, and this preceded the development of a distant focus of infection. This suggests that culture of bone-marrow may be of value in certain blood culture-negative patients with melioidosis.

A prospective comparison of co-amoxiclav and the combination of chloramphenicol, doxycycline, and co-trimoxazole for the oral maintenance treatment of melioidosis.

An open randomized comparison of the oral 'conventional' regimen (combination of chloramphenicol, cotrimoxazole and doxycycline) and co-amoxiclav for the maintenance treatment of melioidosis was conducted in Ubon Ratchatani, north-eastern Thailand, between 1989 and 1992. The total antibiotic treatment duration was 20 weeks. Of 101 patients followed, 10 (10%; 95% confidence interval [CI] 4.9-17.5%) subsequently relapsed: 2 of 52 patients (4%) in the oral 'conventional' group, and 8 of 49 patients (16%) receiving oral co-amoxiclav. This compares with a relapse rate of 23% in our previous study of 8 weeks' total therapy. Only 50% of patients complied with the 20 weeks' treatment regimen and poor compliance proved the most significant risk factor for subsequent relapse (relative risk [RR] 4.9, 95% CI 1.2-20.3). Neither the presence of known underlying disease nor choice of initial parenteral treatment was significantly associated with a higher risk of relapse. Co-amoxiclav is safer and better tolerated, but may be less effective (RR of relapse 0.4, 95% CI 0.2-1.2) than the oral 'conventional' regimen. The minimum duration of total treatment with either regimen should be 12-20 weeks, depending on clinical progress.

The changing pattern of bloodstream infections associated with the rise in HIV prevalence in northeastern Thailand.

A survey of bloodstream infections was conducted in the large regional hospital in Ubon Ratchatani, northeastern Thailand between 1989 and 1998, during the onset of the HIV epidemic. The incidence of Staphylococcus aureus, Escherichia coli, Klebsiella/Enterobacter and Pseudomonas aeruginosa bacteraemias remained constant whereas infections caused by Burkholderia pseudomallei, non-typhoid Salmonellae, Cryptococcus neoformans, Penicillum marneffei and to a lesser extent Streptococcus pneumoniae all rose. Burkholderia pseudomallei infections were unrelated to HIV, whereas the other infections were associated directly with HIV. Group D non-typhoid Salmonellae bloodstream infections (mainly Salmonella enteritidis) rose coincident with the increase in HIV seroprevalence, and preceded the increase in the other HIV-associated infections. Other non-typhoid Salmonella bacteraemias increased two years after the rise in group D infections, and invasive yeast infections increased four years later, coincident with the increase in AIDS. Increasing Group D non-typhoid Salmonella bloodstream infections are an early warning signal of an impending rise in AIDS.

Recent advances in the treatment of severe melioidosis.

For the last decade, high-dose intravenous ceftazidime has been the drug of choice for the treatment of severe melioidosis, after ceftazidime was shown to be superior to the 'conventional' four-drug regimen (chloramphenicol, doxycycline and trimethoprim-sulphamethoxazole) in a randomised trial. Combination ceftazidime-trimethoprim-sulphamethoxazole was compared with the conventional regimen in a separate trial with similar results, but we still do not know whether such combination therapy is needed in melioidosis. Co-amoxiclav (amoxycillin-clavulanate) has been shown to be effective but was associated with a higher rate of treatment failure than ceftazidime. Two further treatment trials in acute melioidosis have recently been conducted in Thailand. In the first of these, high-dose intravenous imipenem was compared with ceftazidime and the results suggest that the two regimens possess similar efficacy. Cefoperazone-sulbactam has been compared with ceftazidime-trimethoprim-sulphamethoxazole in a small number of patients, and further results are awaited. Relapses of melioidosis should be treated in a similar manner to primary infections.

Glucose tolerance in rural diabetic Thais, first-degree relatives and non-diabetic controls.

To determine whether non-insulin-dependent diabetes mellitus (NIDDM) in a rural Thai population is characterised by insulin resistance and hyperinsulinaemia, 17 unselected diabetic outpatients from a regional hospital, five first-degree relatives and 10 healthy controls were studied. Subjects in these groups were matched as closely as possible for age and sex, and mean body mass indices were similar (mean +/- S.D.; 21.8 +/- 5.5, 20.6 +/- 1.4 and 21.8 +/- 2.3 kg/m2, respectively, P > 0.5). Beta-cell function (%B) and insulin sensitivity (%S), expressed relative to values for non-diabetic Caucasians, were assessed mathematically using the 'CIGMA' model and plasma glucose and insulin achieved after a standard 1-h glucose infusion. The diabetic patients had higher fasting plasma glucose concentrations than the controls (8.6 +/- 4.0 vs. 4.6 +/- 0.4 mmol/l, P < 0.01) but plasma insulin levels were comparable (geometric mean [-S.D.-+S.D.]; 4.0 [1.7-9.4] vs. 4.0 [1.7-9.2] mU/l, P > 0.1). %B in the diabetic group (21% [10-41]) was lower than in the controls (128% [88-187], P < 0.001) while %S tended to be higher (185% [86-400] vs. 111% [49-251], 0.1 > P > 0.05). Relatives had intermediate values of both variables. %S and %B correlated poorly in the diabetic group (P > 0.1) but together accounted for 90% of the variation in basal plasma glucose (multiple r = 0.95, n = 17, P < 0.0001). Beta-cell dysfunction appears the primary defect in diabetic patients from a Thai subsistence farming population. Insulin resistance may not always characterise NIDDM in geographical areas where a 'thrifty genotype' would be expected; other factors associated with diabetes in developing countries (such increased susceptibility to serious infections) may also influence diabetes prevalence.

Passive protection of diabetic rats with antisera specific for the polysaccharide portion of the lipopolysaccharide isolated from Pseudomonas pseudomallei.

Polyclonal and monoclonal antisera raised to tetanus toxoid-conjugated polysaccharide of lipopolysaccharide (lps) and purified lps of Pseudomonas pseudomallei that reacted with a collection of 41 strains of this bacterium from 23 patients are described. The common antigen recognized by these sera was within the polysaccharide component of the lps of the cells. The sera were specific for P pseudomallei in that none of 37 strains of other bacteria, including 20 Gram-negative and three Gram-positive species, were recognized, although cross-reaction occurred using the anticonjugate serum with some strains of Pseudomonas cepacia serotype A, a closely related bacterium. Passive protection studies using a diabetic rat model of P pseudomallei infection showed that partially purified rabbit polyclonal and mouse monoclonal antisera were protective when the median lethal dose was raised by four to five orders of magnitude. The wide distribution of the polysaccharide antigen among isolates of P pseudomallei used in this study and the protective role of antibody to the conjugated polysaccharide antigen suggest potential as a vaccine.

Melioidosis; a treatment challenge.

Ceftazidime has reduced the mortality of severe disease by half, but melioidosis remains a difficult and expensive infection to treat. Empirical treatment of septicemia with aminoglycosides combined with penicillin, ampicillin, or second-generation cephalosporins is ineffective. The response to appropriate antibiotic treatment is slow, and most patients require a minimum of 2 weeks of high-dose parenteral treatment. Large abscesses should be drained if possible. Ceftazidime remains the drug of choice, but co-amoxyclav is an effective alternative (although treatment failure rates are slightly higher), and preliminary experience with imipenem is encouraging. The relapse rate following 8 weeks of treatment is approximately 28%, and this is reduced to 9% with 20 weeks of treatment. The relapse rate is determined by the extent of the infection and not the underlying predisposing condition. Resistance to all treatment antimicrobials has been documented, but this has not proved a major problem to date. Patients who survive the acute phase of melioidosis require life-long follow-up.

Siderophore production by Pseudomonas pseudomallei.

Eighty-four strains of Pseudomonas pseudomallei isolated from patients with melioidosis were examined for siderophore production. All the strains were shown to produce siderophore both on chrome azurol S agar plates and in liquid medium under iron-deficient conditions. Chemical assays indicated that the siderophore belongs to the hydroxamate class. Addition of iron to the culture medium resulted in increased culture growth with markedly decreased yield of siderophore. Siderophore produced by strain U7 was purified by gel filtration chromatography, and the molecular weight was estimated to be 1,000. When this partially purified siderophore was added to culture medium, it promoted iron uptake by P. pseudomallei in the presence of EDTA and enhanced growth of the organism in the presence of transferrin. We have given this siderophore the trivial name malleobactin.