Rapid Immunofluorescence Microscopy for Diagnosis of Melioidosis

An immunofluorescent (IF) method that detects Burkholderia pseudomallei in clinical specimens within 10 min was devised. The results of this rapid method and those of an existing IF method were prospectively compared with the culture results for 776 specimens from patients with suspected melioidosis. The sensitivities of both IF tests were 66%, and the specificities were 99.5 and 99.4%, respectively.     

Comparison of Ashdown's medium, Burkholderia cepacia medium, and Burkholderia pseudomallei selective agar for clinical isolation of Burkholderia pseudomallei

Ashdown's medium, Burkholderia pseudomallei selective agar (BPSA), and a commercial Burkholderia cepacia medium were compared for their abilities to grow B. pseudomallei from 155 clinical specimens that proved positive for this organism. The sensitivity of each was equivalent; the selectivity of BPSA was lower than that of Ashdown's or B. cepacia medium.     

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.     

Failure of Burkholderia pseudomallei to Grow in an Automated Blood Culture System

We compared the organisms isolated from 30,210 pairs of blood culture bottles by using BacT/Alert system and the conventional system. Overall, 2,575 (8.5%) specimens were culture positive for pathogenic organisms. The sensitivity for detection of pathogenic organisms with the BACT/Alert system (85.6%, 2,203 of 2,575) was significantly higher than that with the conventional method (74.1%, 1,908 of 2,575; P < 0.0001). However, Burkholderia pseudomallei was isolated less often with the BacT/ALERT system (73.5%, 328 of 446) than with the conventional system (90.3%, 403 of 446; P < 0.0001). This finding suggests that use of the conventional culture method in conjunction with the BacT/Alert system may improve the isolation rate for B. pseudomallei in melioidosis-endemic areas.The Gram-negative bacillus Burkholderia pseudomallei is a Tier 1 select agent and the cause of melioidosis.¹ The disease accounts for 20% of all community-acquired septicemias in northeastern Thailand,² where melioidosis is the third most frequent cause of death from infectious diseases.³ Melioidosis is notoriously difficult to cure despite appropriate antimicrobial therapy and has a case-fatality rate of up to 43%.³ More than half of all melioidosis patients are bacteremic, and positive blood cultures for B. pseudomallei obtained at hospital admission and/or during hospitalization are strong prognostic markers for death.⁴Although the automated blood culture system (BacT/Alert) is convenient and currently used in many laboratories in provincial hospitals in Thailand, it is unclear whether its sensitivity for the detection of pathogens is similar to that obtained using a conventional low tech system still commonly used in small hospitals in resource-limited settings.In a retrospective study conducted during January 1, 2009–July 31, 2011 as part of routine patient care at Sappasithiprasong Hospital, a 1,000-bed tertiary-care hospital in northeastern Thailand, we compared the organisms isolated from more than 30,000 pairs of blood culture bottles by using the BacT/Alert system and the conventional system.In conventional system, the culture medium is made in-house, blood culture bottles are incubated in a conventional incubator, and bacterial detection is made by direct visualization with or without regular sub-culture. During the study period, two 5-mL blood samples were regularly obtained from each patient 10–15 minutes apart. The first 5 mL of blood was inoculated into a 40 mL culture media BacT/Alert SA bottle (catalog no. 259789; bioMérieux, Durham, NC). The second 5 mL of blood was inoculated into an in-house bottle containing 40 mL of broth, which consisted of 37 g of brain heart infusion medium broth (catalogue no, 211059; Becton Dickinson, Franklin Lakes, NJ) and 0.25 g of sodium polyanatholesulfonate (catalog no.1000907362; Sigma, St. Louis, MO), in 1 liter of purified water. BacT/Alert bottles were incubated in the BacT/Alert automated blood culture system (bioMérieux) at 35°C for 7 days, and in-house bottles were incubated aerobically in a normal incubator at 35°C for 7 days. Examination of BacT/Alert bottles was done according to the directions provided by the manufacturer, and positive cultures were indicated on the computer screen accompanied by a beeping sound. Positive cultures in the in-house bottles were detected by direct visualization of cloudy broth.Positive bottles from both systems were sub-cultured by using airway needles (bioMérieux) to place approximately 15–20 μL of the culture on chocolate agar, blood agar, and eosin-methylene blue agar. In addition, all in-house bottles were routinely sub-cultured onto blood agar on day 2 of incubation, and all in-house and BacT/Alert bottles were routinely sub-cultured onto blood agar on day 7 of incubation. Blood agar and eosin-methylene blue agar plates were incubated aerobically at 35°C and inspected at 24 hours. Chocolate agar plates were incubated in an atmosphere of 5% CO₂ at 35°C and were inspected at 48 hours. Bacterial or fungal colonies that grew on culture plates were identified by using standard biochemical tests and colonies of presumptive B. pseudomallei were identified by typical colony morphology on Ashdown agar, resistance to gentamicin and colistin, and a positive result for a highly specific latex agglutination test, as described.^5,6A total of 30,210 pairs of blood culture bottles were collected during the study period (10,208, 12,574 and 7,428 in 2009, 2010 and 2011, respectively). Overall, 2,575 (8.5%) specimens were culture positive for pathogenic organisms. A total of 1,536 (59.7%) grew with both methods, 667 (25.9%) grew only with the BacT/Alert system, and 372 (14.4%) grew only with the conventional method.The pathogenic organisms isolated were gram-negative bacteria (68.1%), gram-positive bacteria (20.0%), fungi (9.7%) and polymicrobial organisms (2.2%). The most common pathogens were Escherichia coli (18.9%, 486 of 2,575), B. pseudomallei (17.3%, 446 of 2,575), Klebsiella species (10.3%, 266 of 2,575), Staphylococcus aureus (8.5%, 219 of 2,575), and Pseudomonas spp. (8.0%, 207 of 2,575) (

Burkholderia pseudomallei detection in surface water in southern Laos using Moore's swabs

The causal agent of melioidosis, Burkholderia pseudomallei, has been cultured from paddy fields in the Lao PDR. We carried out a pilot study to examine the relationship between bacterial soil contamination and that of nearby surface waters in Saravane Province. Soil sampling was conducted at a depth of 30 cm (100 holes in a 45 × 45 m grid) at two sites, East and West Saravane. Moore's swabs were used for water sampling of paddy fields, lakes, rivers, boreholes, and storage tanks within 2 km of the two soil sampling sites. B. pseudomallei from soil and water were cultured on Ashdown's agar. Thirty-six percent and 6% of water samples collected around East and West Saravane, respectively, were culture positive for B. pseudomallei. Low pH and high turbidity were independently associated with culture of B. pseudomallei. Most positive water samples were from the Sedone River, downstream of the East Saravane site. Moore's swabs are simple and inexpensive tools for detecting B. pseudomallei in surface waters.     

Microevolution of Burkholderia pseudomallei during an Acute Infection

We used whole-genome sequencing to evaluate 69 independent colonies of Burkholderia pseudomallei isolated from seven body sites of a patient with acute disseminated melioidosis. Fourteen closely related genotypes were found, providing evidence for the rapid in vivo diversification of B. pseudomallei after inoculation and systemic spread.     

Pharmacokinetic-pharmacodynamic evaluation of ceftazidime continuous infusion vs intermittent bolus injection in septicaemic melioidosis

AIMS: Experimental studies have suggested that constant intravenous infusion would be preferable to conventional intermittent bolus administration of beta-lactam antibiotics for serious Gram-negative infections. Severe melioidosis (Burkholderia pseudomallei infection) carries a mortality of 40% despite treatment with high dose ceftazidime. The aim of this study was to measure the pharmacokinetic and pharmacodynamic effects of continuous infusion of ceftazidime vs intermittent bolus dosing in septicaemic melioidosis. METHODS: Patients with suspected septicaemic melioidosis were randomised to receive ceftazidime 40 mg kg-1 8 hourly by bolus injection or 4 mg kg-1 h-1 by constant infusion following a 12 mg kg-1 priming dose to perform estimation of pharmacokinetic and pharmacodynamic parameters. RESULTS: Of the 34 patients studied 16 (59%) died. Twenty patients had cultures positive for B. pseudomallei of whom 12 (60%) died. The median MIC90 of B. pseudomallei was 2 mg l-1, giving a target concentration CT, of 8 mg l-1. The median (range) estimated total apparent volume of distribution, systemic clearance and terminal elimination half-lives of ceftazidime were 0.468 (0.241-0.573) l kg-1, 0.058 (0.005-0.159) l kg-1 h-1 and 7.74 (1.95-44.71) h, respectively. Clearance of ceftazidime and creatinine clearance were correlated closely (r = 0. 71; P < 0.001) and there was no evidence of significant nonrenal clearance. CONCLUSIONS: Simulations based on these data and the ceftazidime sensitivity of the B. pseudomallei isolates indicated that administration by constant infusion would allow significant dose reduction and cost saving. With conventional 8 h intermittent dosing to patients with normal renal function, plasma ceftazidime concentrations could fall below the target concentration but this would be unlikely with a constant infusion. Correction for renal failure which is common in these patients is Clearance = k * creatinine clearance where k = 0.072. Calculation of a loading dose gives median (range) values of loading dose, DL of 3.7 mg kg-1 (1. 9-4.6) and infusion rate I = 0.46 mg kg h-1 (0.04-1.3) (which equals 14.8 mg kg-1 day-1). A nomogram for adjustment in renal failure is given.     

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.