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1 JCM Accepts, published online ahead of print on 26 March 2014 J. Clin. Microbiol. doi: /jcm Copyright 2014, American Society for Microbiology. All Rights Reserved. 1 2 Evaluation of the Remel RapID NF Plus Rapid Biochemical Method for the Identification of Burkholderia pseudomallei Samuel Maloney, a, b# Cathy Engler, b Robert Norton. a,b The Townsville Hospital, Townsville, Queensland, Australia; Microbiology, Pathology Queensland, Townsville, Queensland, Australia Running Head: RAPID identification of Burkholderia pseudomallei. # Address correspondence to Samuel Maloney, sam.maloney@dhhs.tas.gov.au Downloaded from on September 16, 2018 by guest

2 Abstract Typical methods for the identification of Burkholderia pseudomallei colonies produce results in 18 hours. The Remel RapID NF Plus kit produces results in 4 hours. Using the kit we performed identification of 190 stored B. pseudomallei isolates and correctly identified 189. This kit produces consistent results for known B. pseudomallei isolates. Body Burkholderia pseudomallei is the causative agent of melioidosis (1). Diagnosis of this disease in endemic areas primarily relies on its recovery in culture from an infected site and subsequent phenotypic and biochemical identification (1). In areas where this organism is endemic the identity of an isolate is usually suspected from its antibiotic susceptibility profile (gentamicin resistant, amoxicillin/clavulanic acid sensitive) and characteristic colony types (2), this presumptive identification is usually confirmed on automated systems (such as the VITEK 2 (biomérieux)) which takes up to18 hours, or the API 20 NE or API 20 E (biomérieux) which take hours (2, 3). Updates to the VITEK 2 database have decreased misidentification rates and has increased the reliability of this approach (4). Misidentification by these systems has been reported (5), and the reliability of this system is regionally dependent (6). The RapID NF Plus is a biochemical panel of tests that consists of 3 unifunctional and 7 bifunctional wells, for a total of 17 results. The interpretation of these strips requires the gram stain and oxidase result to be known and results are available in 4 hours. RapID NF Plus (Thermo Fisher Scientific), has been previously reported to be unreliable for the identification of B. pseudomallei.(7) In August 2013 an update of the database

3 (ERIC{trade mark, serif} Electronic Compendium (version , Patch 0613)) added profiles for the identification of B. pseudomallei by the RapID NF Plus kit. A reduction in the time to identification of this organism has the potential to alter patient management in both endemic and non-endemic areas. We investigated the reliability of his method by performing RapID NF Plus identification on 190 stored isolates of B. pseudomallei. Clinical strains were taken from a local collection stored on latex beads in a -70 C freezer. Most isolates come from the Townsville region. Strains had been previously identified by a combination of phenotypic tests, VITEK, API 20NE and most also serotyped. The clinical cases that were drawn from were not related. Strains were thawed and inoculated onto horse blood agar (HBA) plates. These plates were incubated aerobically at 36 C for 18 hours, before single colonies of the isolates were subbed onto another HBA plate and incubated for a further 24 hours. These plates were inspected visually for purity and morphology, and an oxidase reaction was performed. Each isolate then had a RapID NF Plus test performed, as per the manufacturer s instructions. Briefly, single or representative colonies were inoculated into the RapID NF Plus inoculation fluid. This was then transferred into a RapID NF Plus strip and subsequently distributed into the reaction cavities. The strips were incubated at 36 C for 4 hours. After incubation, the strips were interpreted as per instructions, including the addition of specific reagents to the specified wells. Interpreted results were entered into the electronic RapID code compendium (ERIC {trade mark, serif} Electronic Compendium (version , Patch 0613) and microcode corresponding to a specific identification was assigned. RapID NF Plus identified our previously characterised isolates as B. pseudomallei as the first or only option in 99.47% of cases (table 1). This compares well to published results from other systems of B. pseudomallei identification (table 2). The one failed ID has been

4 previously characterised by API 20NE, VITEK and serotyped. The stored isolate was retrieved after completion of the study and confirmed as B. pseudomallei by VITEK II ID and by qrt PCR using a previously characterised method. (11) The RapID NF Plus with ERIC {trade mark, serif} Electronic Compendium (version , Patch 0613) provides an alternative method for identifying colonies suspected to be B. pseudomallei. This may decrease time to diagnosis and allow more a rapid switch to appropriate therapy and provide an alternative means of identification of this critical pathogen in Northern Australia. Further validation will need to be performed to confirm this finding on a geographically diverse collection of isolates. Acknowledgements RapID NF Plus kits were provided by Thermo Fisher Scientific. 1. Wiersinga WJ, Currie BJ, Peacock SJ Melioidosis. N Engl J Med 367: Dance DA, Wuthiekanun V, Naigowit P, White NJ Identification of Pseudomonas pseudomallei in clinical practice: use of simple screening tests and API 20NE. J Clin Pathol 42: Lowe P, Engler C, Norton R Comparison of automated and nonautomated systems for identification of Burkholderia pseudomallei. J Clin Microbiol 40: Lowe P, Haswell H, Lewis K Use of various common isolation media to evaluate the new VITEK 2 colorimetric GN Card for identification of Burkholderia pseudomallei. J Clin Microbiol 44:

5 Zong Z, Wang X, Deng Y, Zhou T Misidentification of Burkholderia pseudomallei as Burkholderia cepacia by the VITEK 2 system. J Med Microbiol 61: Podin Y, Kaestli M, McMahon N, Hennessy J, Ngian HU, Wong JS, Mohana A, Wong SC, William T, Mayo M, Baird RW, Currie BJ Reliability of automated biochemical identification of Burkholderia pseudomallei is regionally dependent. J Clin Microbiol 51: Glass MB, Popovic T Preliminary evaluation of the API 20NE and RapID NF plus systems for rapid identification of Burkholderia pseudomallei and B. mallei. J Clin Microbiol 43: Inglis TJ, Merritt A, Chidlow G, Aravena-Roman M, Harnett G Comparison of diagnostic laboratory methods for identification of Burkholderia pseudomallei. J Clin Microbiol 43: Amornchai P, Chierakul W, Wuthiekanun V, Mahakhunkijcharoen Y, Phetsouvanh R, Currie BJ, Newton PN, van Vinh Chau N, Wongratanacheewin S, Day NP, Peacock SJ Accuracy of Burkholderia pseudomallei identification using the API 20NE system and a latex agglutination test. J Clin Microbiol 45: Koh TH, Yong Ng LS, Foon Ho JL, Sng LH, Wang GC, Tzer Pin Lin RV Automated identification systems and Burkholderia pseudomallei. J Clin Microbiol 41: Hodgson K, Engler C, Govan B, Ketheesan N, Norton R Comparison of Routine Bench and Molecular Diagnostic Methods in Identification of Burkholderia pseudomallei. J Clin Microbiol 47:

6 Table 1: The RapID NF Plus with ERIC{trade mark, serif} Electronic Compendium (version ) Patch 0613). Microcode Instances Identification B. pseudomallei 93.5% Shewanella putrefaciens 6.5% B. pseudomallei 99.12% Burkholderia cepacia 0.88% B. pseudomallei 99.9% B. pseudomallei 99.9% B. pseudomallei 99.9% B. pseudomallei 94.42% Shewanella putrefaciens 5.57% Failed ID Total correct ID 189 Total isolates 190 Correct 99.47% Table 2: Comparison of commercially available and automated biochemical systems Method Correct ID Region Year Ref API 20 NE 99.75% Australia 1989 (2) 98% Australia 2002 (3) 60% Diverse 2005 (7) 37% Diverse 2005 (8) 99% Thailand/Australia 2007 (9)

7 118 API 20 E 99% Australia 2002 (3) RapID NF Plus 0% Diverse 2005 (7) Vitek 1 99% Australia 2002 (3) Vitek 2 19% Australia 2002 (3) 99% Australia 2006 (4) 53-98% Malaysia and 2013 (6) Australia BD Phoenix 0% Singapore 2003 (10) Downloaded from on September 16, 2018 by guest