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.     

Increasing Incidence of Human Melioidosis in Northeast Thailand

Melioidosis is a serious community-acquired infectious disease caused by the Gram-negative environmental bacterium Burkholderia pseudomallei. A prospective cohort study identified 2,243 patients admitted to Sappasithiprasong Hospital in northeast Thailand with culture-confirmed melioidosis between 1997 and 2006. These data were used to calculate an average incidence rate for the province of 12.7 cases of melioidosis per 100,000 people per year. Incidence increased incrementally from 8.0 (95% confidence interval [CI] = 7.2–10.0) in 2000 to 21.3 (95% CI = 19.2–23.6) in 2006 (P < 0.001; χ² test for trend). Male sex, age ≥ 45 years, and either known or undiagnosed diabetes were independent risk factors for melioidosis. The average mortality rate from melioidosis over the study period was 42.6%. The minimum estimated population mortality rate from melioidosis in 2006 was 8.63 per 100,000 people (95% CI = 7.33–10.11), the third most common cause of death from infectious diseases in northeast Thailand after human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) and tuberculosis.     

Simultaneous infection with more than one strain of Burkholderia pseudomallei is uncommon in human melioidosis

A prospective study was performed to determine the rate at which patients with melioidosis are infected with more than one strain of Burkholderia pseudomallei. Genotyping of 2,058 bacterial colonies isolated from 215 samples taken from 133 patients demonstrated that mixed infection is uncommon (2/133 cases [1.5%; 95% confidence interval, 0.2 to 5.3%]).     

Colony Morphology Variation of Burkholderia pseudomallei Is Associated with Antigenic Variation and O-Polysaccharide Modification

Burkholderia pseudomallei is a CDC tier 1 select agent that causes melioidosis, a severe disease in humans and animals. Persistent infections are common, and there is currently no vaccine available. Lipopolysaccharide (LPS) is a potential vaccine candidate. B. pseudomallei expresses three serologically distinct LPS types. The predominant O-polysaccharide (OPS) is an unbranched heteropolymer with repeating d-glucose and 6-deoxy-l-talose residues in which the 6-deoxy-l-talose residues are variably replaced with O-acetyl and O-methyl modifications. We observed that primary clinical B. pseudomallei isolates with mucoid and nonmucoid colony morphologies from the same sample expressed different antigenic types distinguishable using an LPS-specific monoclonal antibody (MAb). MAb-reactive (nonmucoid) and nonreactive (mucoid) strains from the same patient exhibited identical LPS banding patterns by silver staining and indistinguishable genotypes. We hypothesized that LPS antigenic variation reflected modification of the OPS moieties. Mutagenesis of three genes involved in LPS synthesis was performed in B. pseudomallei K96243. Loss of MAb reactivity was observed in both wbiA (encoding a 2-O-acetyltransferase) and wbiD (putative methyl transferase) mutants. The structural characteristics of the OPS moieties from isogenic nonmucoid strain 4095a and mucoid strain 4095c were further investigated. Utilizing nuclear magnetic resonance (NMR) spectroscopy, we found that B. pseudomallei 4095a and 4095c OPS antigens exhibited substitution patterns that differed from the prototypic OPS structure. Specifically, 4095a lacked 4-O-acetylation, while 4095c lacked both 4-O-acetylation and 2-O-methylation. Our studies indicate that B. pseudomallei OPS undergoes antigenic variation and suggest that the 9D5 MAb recognizes a conformational epitope that is influenced by both O-acetyl and O-methyl substitution patterns.     

Quantitation of B. Pseudomallei in clinical samples

We undertook a prospective study to quantitate Burkholderia pseudomallei in blood, urine, respiratory secretions, and pus [corrected] obtained from 414 patients with melioidosis. The median was count 1.1, 1.5 x 10(4), 1.1 x 10(5), and 1.1 x 10(7) CFU/mL in these sample types, respectively. This provides important insights into the likely feasibility of future studies such as expression microarray analysis using clinical material.     

Prospective evaluation of a rapid immunochromogenic cassette test for the diagnosis of melioidosis in northeast Thailand

A prospective study was performed to compare a rapid immunochromogenic cassette test (ICT) with the indirect haemagglutination assay (IHA) and clinical rules for the diagnosis of melioidosis in an endemic area. The sensitivity and specificity of the IgG ICT was 86% and 47%, and the IgM ICT was 82% and 47%, respectively. These were similar to the results for IHA (sensitivity 73%, specificity 64%) and clinical rules (73% and 37%). ICT lacks clinical utility as a result of high background rates of positive Burkholderia pseudomallei serology in this population. Low sensitivity and specificity of clinical rules is consistent with the protean manifestations of melioidosis and clinical difficulty in identifying patients with melioidosis.     

Melioidosis Caused by Burkholderia pseudomallei in Drinking Water,Thailand, 2012

We identified 10 patients in Thailand with culture-confirmed melioidosis who had Burkholderia pseudomallei isolated from their drinking water. The multilocus sequence type of B. pseudomallei from clinical specimens and water samples were identical for 2 patients. This finding suggests that drinking water is a preventable source of B. pseudomallei infection.     

Monoclonal Antibody-Based Immunofluorescence Microscopy for the Rapid Identification of Burkholderia pseudomallei in Clinical Specimens

The diagnosis of melioidosis depends on the culture of Burkholderia pseudomallei, which takes at least 48 hours. We used a polyclonal-FITC-based immunofluorescence microscopic assay (Pab-IFA) on clinical samples to provide a rapid presumptive diagnosis. This has limitations including photobleaching and batch-to-batch variability. This study evaluated an IFA based on a monoclonal antibody specific to B. pseudomallei (Mab-IFA) and Alexa Fluor 488. A diagnostic evaluation was performed on a prospective cohort of 951 consecutive patients with suspected melioidosis. A total of 1,407 samples were tested. Test accuracy was defined against culture as the gold standard, and was also compared against Pab-IFA. A total of 88 samples from 64 patients were culture positive for B. pseudomallei. The diagnostic sensitivity and specificity of the Mab-IFA was comparable to the Pab-IFA (48.4% versus 45.3% for sensitivity, and 99.8% versus 98.8% for specificity). We have incorporated the Mab-IFA into our routine practice.Melioidosis is an infectious disease caused by the environmental Gram-negative bacterium, Burkholderia pseudomallei. Most cases are reported from northern Australia and southeast Asia, and infection results from bacterial inoculation, inhalation, or ingestion.¹ In northeast Thailand, melioidosis is the third most common cause of death from an infectious disease, after human immunodeficiency virus (HIV) infection and tuberculosis.² Clinical features of melioidosis are diverse, but the most common manifestations are septicemia (50% of cases), pneumonia, and abscesses in internal organs.³ Mortality in Thailand is 40%, rising to 90% in those with severe sepsis. Rapid diagnosis and administration of effective antimicrobial therapy is life saving, because B. pseudomallei is inherently resistant to a range of antibiotic classes, and patients require ceftazidime or a carbapenem drug.^1,3,4 Laboratory diagnosis is culture-based, which takes at least 48 hours from sample receipt to confirmed identification. Molecular methods including real-time polymerase chain reaction (PCR) and loop-isothermal amplification have been described, which can be applied to DNA extracted directly from the clinical sample, and these can provide a more rapid diagnosis but have a lower diagnostic sensitivity than culture.^5,6Our clinical research laboratory in Sappasithiprasong Hospital, Ubon Ratchathani, northeast Thailand has used an in-house immunofluorescence microscopy assay (IFA) for the rapid detection of B. pseudomallei in clinical specimens since 1993.⁷ This uses a fluorescein isothiocyanate (FITC)-labeled rabbit polyclonal antibody (Pab) against formalin-killed B. pseudomallei, two clinical evaluations have reported a sensitivity and specificity of 66–73% and 99%, respectively.^7,8 Used as an adjunct to culture, this can provide a very rapid presumptive diagnosis of melioidosis, pending culture confirmation. This assay has several limitations, however, including photobleaching of FITC^9–11 and the potential for higher batch-to-batch variation with polyclonal compared with monoclonal antibody (Mab) preparations.^12,13 We have produced an in-house Mab that recognizes B. pseudomallei exopolysaccharide,¹⁴ and the aim of this study was to re-evaluate the IFA in the laboratory and clinical settings after replacing the Pab with this Mab and replacing FITC with a photo-stable dye.The Mab-IFA was developed as an indirect assay. The primary detection antibody was unlabelled Mab 4B11 (IgG2b subclass) specific to B. pseudomallei exopolysaccharide,¹⁴ and the secondary antibody was Alexa Fluor 488 conjugated-goat anti-mouse immunoglobulin G (IgG) (Molecular Probes, Carlsbad, CA). Mab was prepared from culture supernatant of hybridroma clone 4B11, as described previously.¹⁴ The Mab-IFA was optimized for ease of use by preparing a single mixture of primary and secondary antibody, which was added to the slide in a single step. The Mab-IFA detection reagent contained 5 μg/mL of Mab and 20 μg/mL of secondary antibody in phosphate-buffered saline (PBS). The limit of detection was defined using a 10-fold dilution series ranging from 2×10¹⁰ to 20 colony-forming units (CFU)/mL of B. pseudomallei K96243. Ten microliters of each bacterial dilution was mixed with an equal volume of Mab-IFA and incubated at room temperature for 5 min before observing for the presence of green fluorescent bacteria using a fluorescent microscope at 1,000× magnification (Olympus BH-2, Tokyo, Japan). Burkholderia pseudomallei appeared as individual, uniformly stained bacilli (Figure 1​1AA and ​andB).B). The limit of detection of the assay, defined as the lowest number of bacteria that gave a positive result for Mab-IFA, was 2×10³ CFU/mL.Open in a separate windowFigure 1.Fluorescent microscopy of Burkholderia pseudomallei stained with Mab-IFA reagent. The bacteria shown were from laboratory cultures on Columbia agar (A) or LB broth (B), or from clinical samples (urine [C], pus [D], or sputum [E]) from patients with melioidosis. The atypical appearance of the bacterial morphology including bacterial elongation (D and E) and swollen cells (C) was not uncommon.The assay sensitivity of the Mab-IFA was defined using 20 clinical B. pseudomallei isolates. The Mab-IFA assay specificity was defined by testing 160 microorganisms representing a wide range of species. These were 20 Gram-positive bacteria (Staphylococcus aureus [16], unknown species of α-hemolytic Streptococcus [1], Streptococcus pneumoniae, [2], Nocardia spp. [1]), 136 Gram-negative bacteria (Acinetobacter spp. [5], A. baumannii [5], Burkholderia thailandensis [10]), Chromobacterium violaceum [1], Haemophilus influenzae [1], Achromobacter xylosoxidans [15]), Aeromonas spp. [22], Burkholderia cepacia [7], Burkholderia pickettii [2], Citrobacter spp. [1], C. freundii [2], Enterobacter spp. [5], E. cloacae [1], Escherichia coli [1], E. fergusonii [1), Flavobacterium spp. [1], Hafnia alvei [1), Klebsiella spp. [2], K. aerogenes [2], K. oxytoca [3], K. ozaenae [1], K. pneumoniae [7], Morganella morganii [2], Proteus mirabilis [3], Pseudomonas spp. [1], P. acidovorans [3], P. aeruginosa [8], P. alcaligenes [2], P. fluorescens [2], P. mesophilia [1], P. pseudoalcaligenes [2], P. putida [3], P. stuartii [1], P. stutzeri [4], Ralstonia paucula [1], Salmonella enterica serovar Paratyphi A [1], S. enterica serovar Typhi [1], Serratia marcescens [1], Sphingomonas paucimobilis [1], Stenotrophomonas maltophilia [3]), and 4 fungi (Candida spp. [1], Cryptococcus neoformans [3]). Microorganisms were sub-cultured on Columbia agar and incubated overnight at 37°C in air. Fastidious bacteria were sub-cultured on chocolate agar and incubated overnight at 37°C in 5% CO₂. The assay sensitivity was 100% (20 of 20 B. pseudomallei positive), and specificity was 90.0% (144 of 160 other species negative). The 16 false positive tests were all S. aureus, which on immunofluroresence appeared as weakly staining cocci in clusters. The false positive reaction occurred in both the presence and absence of Mab, indicating direct binding of the goat anti-mouse IgG conjugated with Alexa Fluor 488 to the organism. This is likely to be caused by antibody binding to the surface-expressed S. aureus immunoglobulin binding protein, Spa.A diagnostic evaluation of the Mab-IFA was performed on a prospective cohort of 951 patients recruited at Sappasithiprasong Hospital, Ubon Rachanthani, northeast Thailand. These were consecutive patients presenting with suspected melioidosis between May and September 2012, from whom a total of 1,407 samples were taken for culture (respiratory secretions, N = 406; urine, N = 937; pus, N = 21; other body fluids, N = 43). Blood cultures were also taken from the cohort, but these are not included here because the bacterial load in blood is too low for direct microscopic detection. Ethical approval for the study was obtained from the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University.Clinical samples were prepared for examination by Mab-IFA as follows. Up to 10 mL of urine or other body fluid (or the available sample volume if less was collected) was centrifuged at 3,000 rpm and the supernatant discarded before the sediment was used in the IFA. Pus or respiratory secretions were tested without a sample preparation step. Ten microliters of working IFA reagent containing the primary and secondary antibody probe was placed on a glass slide, to which 10 μL of sediment was added and mixed and a coverslip applied. Slides were left at room temperature for 5 min, and then examined, as before, using a fluorescent microscope.Our current polyclonal antibody-based IFA (Pab-IFA) was performed in parallel on every sample, and was performed as described previously⁸; in brief, Pab-IFA reagent (FITC conjugated-rabbit antibody to formalin-killed B. pseudomallei) was diluted 1:200 in 5% skimmed milk. Ten microliters of reagent was placed onto a glass slide, mixed with 10 μL of sample prepared as described previously, and a coverslip applied. The slide was observed as for the Mab-IFA. All slides were read by two independent technicians. Heat-killed B. pseudomallei and B. thailandensis (1 × 10⁶ CFU/mL in PBS) were used in both assays as positive and negative controls, respectively.Statistical analyses were performed by using STATA/SE version 11.1 (StataCorp., College Station, TX). Performance of the two assays was compared using the exact McNemar test. Diagnostic sensitivity, specificity, positive and negative predictive values of the Mab-IFA, and Pab-IFA assays were defined against the result for bacterial culture and identification.A total of 88 samples from 64 patients were culture positive for B. pseudomallei. The diagnostic sensitivity of the Mab-IFA (48.4%; 31 of 64 patients) was comparable to the Pab-IFA (45.3%; 29 of 64 patients) (P = 0.69). Assay sensitivity varied between specimen types, as shown in