Abstract. Melioidosis is a bacterial infection caused by Burkholderia pseudomallei, a gram-negative saprophytic bacillus. Cases occur sporadically in the Americas with an increasing number of cases observed among people with no travel history to endemic countries. To better understand the incidence of the disease in the Americas, we reviewed the literature, including unpublished cases reported to the Centers for Disease Control and Prevention. Of 120 identified human cases, occurring between 1947 and June 2015, 95 cases (79%) were likely acquired in the Americas; the mortality rate was 39%. Burkholderia pseudomallei appears to be widespread in South, Central, and North America.

Identifying etiologies of acute febrile illnesses (AFI) is challenging due to non-specific presentation and limited availability of diagnostics. Prospective AFI studies provide a methodology to describe the syndrome by age and etiology, findings that can be used to develop case definitions and multiplexed diagnostics to optimize management. We conducted a 3-year prospective AFI study in Puerto Rico. Patients with fever ≤7 days were offered enrollment, and clinical data and specimens were collected at enrollment and upon discharge or follow-up. Blood and oro-nasopharyngeal specimens were tested by RT-PCR and immunodiagnostic methods for infection with dengue viruses (DENV) 1–4, chikungunya virus (CHIKV), influenza A and B viruses (FLU A/B), 12 other respiratory viruses (ORV), enterovirus, Leptospira spp., and Burkholderia pseudomallei. Clinical presentation and laboratory findings of participants infected with DENV were compared to those infected with CHIKV, FLU A/B, and ORV. Clinical predictors of laboratory-positive dengue compared to all other AFI etiologies were determined by age and day post-illness onset (DPO) at presentation. Of 8,996 participants enrolled from May 7, 2012 through May 6, 2015, more than half (54.8%, 4,930) had a pathogen detected. Pathogens most frequently detected were CHIKV (1,635, 18.2%), FLU A/B (1,074, 11.9%), DENV 1–4 (970, 10.8%), and ORV (904, 10.3%). Participants with DENV infection presented later and a higher proportion were hospitalized than those with other diagnoses (46.7% versus 27.3% with ORV, 18.8% with FLU A/B, and 11.2% with CHIKV). Predictors of dengue in participants presenting <3 DPO included leukopenia, thrombocytopenia, headache, eye pain, nausea, and dizziness, while negative predictors were irritability and rhinorrhea. Predictors of dengue in participants presenting 3–5 DPO were leukopenia, thrombocytopenia, facial/neck erythema, nausea, eye pain, signs of poor circulation, and diarrhea; presence of rhinorrhea, cough, and red conjunctiva predicted non-dengue AFI. By enrolling febrile patients at clinical presentation, we identified unbiased predictors of laboratory-positive dengue as compared to other common causes of AFI. These findings can be used to assist in early identification of dengue patients, as well as direct anticipatory guidance and timely initiation of correct clinical management.

Taken together, isolation of B. pseudomallei from a soil sample and high seropositivity among patient contacts suggest at least regional endemicity of melioidosis in Puerto Rico. Increased awareness of melioidosis is needed to enable early case identification and early initiation of appropriate antimicrobial therapy.

The bacterium Burkholderia pseudomallei causes melioidosis, which is mainly associated with tropical areas. We analyzed single-nucleotide polymorphisms (SNPs) among genome sequences from isolates of B. pseudomallei that originated in the Western Hemisphere by comparing them with genome sequences of isolates that originated in the Eastern Hemisphere. Analysis indicated that isolates from the Western Hemisphere form a distinct clade, which supports the hypothesis that these isolates were derived from a constricted seeding event from Africa. Subclades have been resolved that are associated with specific regions within the Western Hemisphere and suggest that isolates might be correlated geographically with cases of melioidosis. One isolate associated with a former World War II prisoner of war was believed to represent illness 62 years after exposure in Southeast Asia. However, analysis suggested the isolate originated in Central or South America.

Cases of melioidosis and glanders are rare in the United States, but the etiologic agents of each disease (Burkholderia pseudomallei and Burkholderia mallei, respectively) are classified as Tier 1 select agents because of concerns about their potential use as bioterrorism agents. A rapid, highly sensitive, and portable assay for clinical laboratories and field use is required. Our laboratory has further evaluated a latex agglutination assay for its ability to identify B. pseudomallei and B. mallei isolates. This assay uses a monoclonal antibody that specifically recognizes the capsular polysaccharide produced by B. pseudomallei and B. mallei, but is absent in closely related Burkholderia species. A total of 110 B. pseudomallei and B. mallei were tested, and 36 closely related Burkholderia species. The latex agglutination assay was positive for 109 of 110 (99.1% sensitivity) B. pseudomallei and B. mallei isolates tested.

Burkholderia pseudomallei Bp1651 is resistant to several classes of antibiotics that are usually effective for treatment of melioidosis, including tetracyclines, sulfonamides, and ␤-lactams such as penicillins (amoxicillin-clavulanic acid), cephalosporins (ceftazidime), and carbapenems (imipenem and meropenem). We sequenced, assembled, and annotated the Bp1651 genome and analyzed the sequence using comparative genomic analyses with susceptible strains, keyword searches of the annotation, publicly available antimicrobial resistance prediction tools, and published reports. More than 100 genes in the Bp1651 sequence were identified as potentially contributing to antimicrobial resistance. Most notably, we identified three previously uncharacterized point mutations in penA, which codes for a class A ␤-lactamase and was previously implicated in resistance to ␤-lactam antibiotics. The mutations result in amino acid changes T147A, D240G, and V261I. When individually introduced into select agent-excluded B. pseudomallei strain Bp82, D240G was found to contribute to ceftazidime resistance and T147A contributed to amoxicillin-clavulanic acid and imipenem resistance. This study provides the first evidence that mutations in penA may alter susceptibility to carbapenems in B. pseudomallei. Another mutation of interest was a point mutation affecting the dihydrofolate reductase gene folA, which likely explains the trimethoprim resistance of this strain. Bp1651 was susceptible to aminoglycosides likely because of a frameshift in the amrB gene, the transporter subunit of the AmrAB-OprA efflux pump. These findings expand the role of penA to include resistance to carbapenems and may assist in the development of molecular diagnostics that predict antimicrobial resistance and provide guidance for treatment of melioidosis.

Burkholderia pseudomallei isolates from the Western Hemisphere are difficult to differentiate from those from regions in which melioidosis is traditionally endemic. We used internal transcribed spacer typing to determine that B. pseudomallei isolates from the Western Hemisphere are consistently type G. Knowledge of this relationship might be useful for epidemiologic investigations.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used for rapid bacterial identification. Studies of Burkholderia pseudomallei identification have involved small isolate numbers drawn from a restricted geographic region. There is a need to expand the reference database and evaluate B. pseudomallei from a wider geographic distribution that more fully captures the extensive genetic diversity of this species. Here, we describe the evaluation of over 650 isolates. Main spectral profiles (MSP) for 26 isolates of B. pseudomallei (N = 5) and other Burkholderia species (N = 21) were added to the Biotyper database. MALDI-TOF MS was then performed on 581 B. pseudomallei, 19 B. mallei, 6 B. thailandensis and 23 isolates representing a range of other bacterial species. B. pseudomallei originated from northeast and east Thailand (N = 524), Laos (N = 12), Cambodia (N = 14), Hong Kong (N = 4) and Australia (N = 27). All 581 B. pseudomallei were correctly identified, with 100% sensitivity and specificity. Accurate identification required a minimum inoculum of 5 x 107 CFU/ml, and identification could be performed on spiked blood cultures after 24 hours of incubation. Comparison between a dendrogram constructed from MALDI-TOF MS main spectrum profiles and a phylogenetic tree based on recA gene sequencing demonstrated that MALDI-TOF MS distinguished between B. pseudomallei and B. mallei, while the recA tree did not. MALDI-TOF MS is an accurate method for the identification of B. pseudomallei, and discriminates between this and other related Burkholderia species.