Abstract
We evaluated the API 20NE and the RapID NF Plus systems with 58 Burkholderia pseudomallei and 23 B. mallei strains for identification of these agents, but neither was reliable for confirmatory identification, with only 0 to 60% strains identified accurately. A greater diversity of strains in the system databases would be beneficial.
Burkholderia pseudomallei and B. mallei are classified as category B biological threat agents due to their potential for aerosol dissemination and severe impact on human health (10). B. pseudomallei, an environmental pathogen causing melioidosis, is endemic in areas of Southeast Asia and Australia. Humans typically become infected through contact with contaminated soil and water. Infection with B. mallei causes glanders, primarily a disease of horses. Eradicated from North America 50 years ago by effective testing, restrictions, and animal slaughter, B. mallei historically infected humans who worked alongside afflicted animals. In recent years, B. mallei laboratory exposure and infection have been reported (2, 11).
Rapid and reliable confirmatory identification of B. pseudomallei and B. mallei is crucial because of their potential public health impact if used as biothreat agents. Since no human vaccine is available, the sole intervention available is the timely administration of appropriate antimicrobial therapy (6). Conventional confirmatory identification of B. pseudomallei and B. mallei presently relies on an extensive set of biochemical tests that may require up to 7 days before results are obtained. Consequently, manual and automated identification systems may offer a rapid alternative, especially in first-line laboratories unequipped to perform molecular approaches such as diagnostic PCR or 16S rRNA gene sequencing. We selected the API (bioMérieux, Hazelwood, Mo.) and RapID (Remel, Lenexa, Kans.) systems because of their common use in first-line diagnostic laboratories. Both systems contain profile codes and are approved for use with B. pseudomallei, but neither contains profile codes or is approved to identify B. mallei. We used a geographically and temporally diverse collection of B. pseudomallei and B. mallei strains to preliminarily assess the potential of the API and RapID systems as stand-alone tools for identification of these species.
Bacterial strains.
Fifty-eight B. pseudomallei and 23 B. mallei strains were selected for their geographical origin and temporal diversity (Table 1). Confirmatory identification for all strains was carried out by standard biochemical testing (13) and 16S rRNA gene sequencing (4). Isolates were stored at −70°C in defibrinated rabbit blood until tested. All work was performed according to the manufacturer's instructions and took place in a biological safety cabinet in a biosafety level 3 environment. Oxidase testing was carried out with Bactidrop oxidase (Remel, Lenexa, Kans.). Prior to testing, all strains were subcultured twice on Trypticase soy agar with 5% defibrinated sheep blood (BBL Microbiology Systems, Cockeysville, Md.) and incubated at 37°C for 18 to 24 h. All tests were performed once, and no retesting or additional testing was performed. Control strains were used as recommended by the manufacturer of each rapid system.
TABLE 1.
Designations of 58 B. pseudomallei and 23 B. mallei isolates used in this study
| Species (no. of strains) | CDC identifier | Other identifier | Origina |
|---|---|---|---|
| B. pseudomallei (58) | 2000032024 | Human, US, 2000 | |
| 2000032025 | Human, US, 2000 | ||
| 2000032026 | India, 1995 | ||
| 2000032027 | US, 1968 | ||
| 2000032028 | Human, US, 2000 | ||
| 2000032029 | Human, US, 1994 | ||
| 2001029240 | Human, US, 2001 | ||
| 2002721090 | Human, US, 1980 | ||
| 2002721096 | Human, US, 1981 | ||
| 2002721102 | Human, US, 1983 | ||
| 2002721103 | Human, Netherlands, 1985 | ||
| 2002721108 | Human, US, 1988 | ||
| 2002721114 | Human, US, 1991 | ||
| 2002721115 | Human, US, 1992 | ||
| 2002721116 | Human, US, 1992 | ||
| 2002721123 | Human, Puerto Rico, 1998 | ||
| 2002721124 | Human, US, 1999 | ||
| 2002721145 | Human, Philippines, 1969 | ||
| 2002721146 | Human, US, 1969 | ||
| 2002721161 | Human, US, 1970 | ||
| 2002721162 | Human, Australia, 1970 | ||
| 2002721166 | Human, US, 1973 | ||
| 2002721171 | Human, Venezuela, 1976 | ||
| 2002721177 | Human, US, 1977 | ||
| 2002721181 | Human, US, 1979 | ||
| 2002721184 | Human, Ecuador, 1962 | ||
| 2002721186 | Human, US 1966 | ||
| 2002721209 | Monkey, US, 1069 | ||
| 2002721617 | NCTC 8016 | Sheep, Australia, 1949 | |
| 2002721618 | Monkey, Philippines, 1990 | ||
| 2002721619 | Monkey, Indonesia, 1990 | ||
| 2002721620 | Horse, France, 1976 | ||
| 2002721622 | Sheep, Australia, 1984 | ||
| 2002721623 | Cow, Australia, 1985 | ||
| 2002721624 | Goat, Australia | ||
| 2002721625 | Environment, Singapore, 1991 | ||
| 2002721626 | Environment, Thailand, 1990 | ||
| 2002721628 | Environment, Madagascar, 1977 | ||
| 2002721629 | Environment, Kenya, 1992 | ||
| 2002721630 | Environment, France, 1976 | ||
| 2002721631 | Environment, Australia | ||
| 2002721632 | Environment, Australia | ||
| 2002721633 | Human, Thailand, 1987 | ||
| 2002721634 | Human, Thailand, 1992 | ||
| 2002721635 | Human, Singapore, 1988 | ||
| 2002721636 | NCTC 10276 | Human, Bangladesh, 1960 | |
| 2002721637 | Human, Pakistan, 1988 | ||
| 2002721638 | Human, Vietnam 1963 | ||
| 2002721639 | Human, Kenya, 1980 | ||
| 2002721640 | Human, Papua New Guinea, 1989 | ||
| 2002721641 | Human, Fiji, 1992 | ||
| 2002721642 | Human, Malaysia | ||
| 2002721646 | Human, Holland, 1999 | ||
| 2002721647 | Human, UK, 1999 | ||
| 2002734325 | Monkey, US, 2003 | ||
| 2003000540 | Human, US, 2002 | ||
| 2003021442 | Human, US, 2003 | ||
| 2003021443 | Human, US, 2003 | ||
| B. mallei (23) | 2000031063 | ATCC15310 | Horse, Hungary, 1961 |
| 2000031064 | India | ||
| 2000031065 | Turkey | ||
| 2000031066 | India | ||
| 2000031304 | Human, US, 2000 | ||
| 2002721273 | US, 1956 | ||
| 2002721274 | US, 1956 | ||
| 2002721275 | ATCC 10399 | Horse, China, 1956 | |
| 2002721276 | US, 1956 | ||
| 2002721277 | US, 1956 | ||
| 2002721278 | Human, US, 1964 | ||
| 2002721279 | Human, US, 1964 | ||
| 2002721280 | France, 1972 | ||
| 2002721648 | ATCC 23344 | Human, China | |
| 2002734299 | NCTC 10229 | Hungary, 1961 | |
| 2002734300 | NCTC 10247 | Turkey, 1960 | |
| 2002734301 | NCTC 10260 | Human, Turkey, 1949 | |
| 2002734302 | Turkey | ||
| 2002734303 | NCTC 3709 | Horse, India, 1932 | |
| 2002734304 | NCTC 10248 | Human, Turkey, 1950 | |
| 2002734305 | NCTC 3708 | Mule, India, 1932 | |
| 2002734306 | NCTC 120 | UK, 1920 | |
| 2002734307 |
The source of the isolate is given when available. US, United States; UK, United Kingdom.
API 20NE.
Each strain was inoculated into 0.85% NaCl, and turbidity was adjusted to 0.5 MacFarland standard (bioMerieux, Hazelwood, Mo.). The inoculum was distributed into test strips which were incubated at 30°C and read at 24 and 48 h. Quality control testing was performed with every test. Biochemical reactions were read as positive or negative, translated into numerical profiles, and interpreted with the manufacturer's software (APILAB Plus update 3.3.3).
RapID NF Plus.
Each strain was inoculated into the RapID inoculation fluid, and turbidity was adjusted to between 1.0 and 3.0 MacFarland standard (Remel, Lenexa, Kans.). Strips were inoculated and read after a 4-h incubation at 37°C. Quality control tests were performed with each test. Reactions were read as positive or negative, translated into a biocode, and interpreted with the IDS Electronic Code Compendium V1.3.97.
B. pseudomallei results with API 20NE.
Thirty-one different profiles were obtained with the API 20NE; 35 (60%) of the 58 B. pseudomallei strains were identified correctly, 18 (31%) were misidentified, and 5 (9%) were classified as not identifiable (Table 2). Adipate, mannose, and mannitol assimilation and gelatin hydrolysis were most frequently associated with incorrect or unidentifiable strains resulting in a number of different numerical profiles. In previous studies, this system was reported to identify 80 to 98% of strains correctly (3, 5, 9), but the B. pseudomallei strains used were primarily from clinical specimens in areas where B. pseudomallei is endemic and so lacked geographical, temporal, and source diversity. In another study testing 114 geographically diverse clinical, environmental, and reference Burkholderia spp. and closely related strains (but no B. pseudomallei), API 20NE correctly identified 77% of strains (12). Our study emphasizes the importance of including a greater diversity of strains in the API 20NE database.
TABLE 2.
Results of testing 58 B. pseudomallei strains by the API 20NE system
| Result (n) | Identity (%) | Profile no. (no. of isolates identified) |
|---|---|---|
| Correct identificationa (35) | >80 | 1156574 (2) |
| 1156575 (3) | ||
| 1156577 (2) | ||
| 1554577 (1) | ||
| 1556535 (1) | ||
| 1556557 (2) | ||
| 1556574 (10) | ||
| 1556575 (5) | ||
| 1556576 (1) | ||
| 1556577 (4) | ||
| <80 | 1056574 (1) | |
| 1056575 (1) | ||
| 1456575 (1) | ||
| 5156575 (1) | ||
| Incorrect identificationa (18) | >80 | 0156574 (1)c |
| 1112444 (1)d | ||
| 1150054 (1)d | ||
| 1150475 (1)e | ||
| 1154574 (2)e | ||
| 1540554 (1)f | ||
| 1554574 (2)e | ||
| <80 | 1146575 (2)c | |
| 1154554 (1)e | ||
| 1454554 (1)f | ||
| 1550554 (2)g | ||
| 1556554 (3)c | ||
| Not identifiableb (5) | NAh | 0554554 |
| 1044576 | ||
| 1446574 | ||
| 1556154 | ||
| 5744554 |
Profile identification response was excellent, very good, good, or acceptable.
Profile indentification response was unacceptable, indeterminate, or invalid or no species indentification could be determined.
Identified as Pseudomonas fluorescens.
Identified as Comamonas testosterone/Pseudomonas alcaligenes.
Identified as Pseudomonas aeruginosa.
Identified as Aeromonas salmonicida.
Identified as Chromobacterium violaceum.
NA, not applicable.
B. pseudomallei results with RapID NF Plus.
None of the 58 B. pseudomallei strains was identified correctly with the RapID NF Plus, 30 (52%) were misidentified, and 28 (48%) were classified as not identifiable by the 13 microcodes obtained (Table 3); tests for arginine hydrolysis, p-Nitrophenyl-N-acetyl-β-d-glucosaminide, and N-nezyl-arginine-β-napthylamide weremost frequently associated with incorrect or nonidentifiable strains. In a previous study, Rapid NF Plus correctly identified 80 to 90% of nonfermenting gram-negative bacilli (7, 8); however, no reports are available to date on use of this system for identification of B. pseudomallei. Kiska et al. (7) tested 150 nonfermenting strains and reported difficulties in using this system to identify members of the genus Burkholderia, concluding that the conventional biochemical identification is still preferable.
TABLE 3.
Results of testing 58 B. pseudomallei strains by the RapID NF Plus system
| Result (n) | Profile no. (no. of isolates identified) |
|---|---|
| Incorrect identificationa (30) | 430014 (4)c |
| 430016 (9)c | |
| 430204 (1)c | |
| 430216 (5)c | |
| 530016 (2)d | |
| 610016 (2)e | |
| 630014 (3)e | |
| 630236 (4)f | |
| Not identifiableb (28) | 510016 (1) |
| 630016 (7) | |
| 630017 (2) | |
| 630216 (14) | |
| 730016 (4) |
Correct identifications registered an excellent, very good, good, implicit, satisfactory, or adequate biocode. Probability, >95%.
Not identifiable biocodes were the result of a probability overlap between two or more possibilities; additional tests were required or gave an incorrect or unidentified response.
Identified as Burkholderia cepacia.
Identified as Chromobacterium violaceum.
Identified as Comamonas testosteroni.
Identified as Shewanella putrefaciens.
Neither system incorporates B. mallei in its diagnostic algorithm, but both use the same biochemical tests commonly used to identify this agent by conventional methods. Consequently, we also evaluated both systems for the ability to confirm B. mallei.
B. mallei results with API 20NE.
Six (26%) of the B. mallei strains were identified as other organisms, and 17 (74%) were not identifiable (Table 4). With 15 profiles generated from 23 strains, this system was unable to present a cohesive identification for B. mallei. However, it shows potential in that the majority of those profiles were not identifiable and would not cause a misidentification if encountered.
TABLE 4.
Results of testing 23 Burkholderia mallei strains by API 20NE
| Result (n) | Identity (%) | Profile no. (no. of isolates identified) |
|---|---|---|
| Incorrect identificationa (6) | >80 | 1040400 (2)c |
| <80 | 1000000 (1)c | |
| 1040404 (1)d | ||
| 1040500 (2)c | ||
| Not identifiableb (17) | NAe | 0040500 (1) |
| 1041500 (1) | ||
| 1042500 (3) | ||
| 1042520 (1) | ||
| 1042521 (1) | ||
| 1044420 (1) | ||
| 1140500 (2) | ||
| 1140504 (1) | ||
| 1140520 (1) | ||
| 1144501 (1) | ||
| 1146520 (4) |
Profile identification response was excellent, very good, good, or acceptable.
Profile indentification response was unacceptable, indeterminate, or invalid or no species indentification could be determined.
Identified as Pasteurella sp.
Identified as Aeromonas salmonicida masoucida/achromogenes.
NA, not applicable.
B. mallei results with rapID NF Plus.
Eleven (48%) of the B. mallei strains were identified as other organisms, and 12 (52%) were not identifiable (Table 5); these 23 strains produced 12 profiles. Microcodes 430012 and 630012 were the most commonly identified.
TABLE 5.
Results of testing 23 Burkholderia mallei strains by RapID NF Plus
| Resulta (n) | Probability (%) | Profile no. (no. of isolates identified) |
|---|---|---|
| Incorrect identification (11) | >95 | 030010 (1)c |
| 400012 (1)c | ||
| 410002 (1)d | ||
| 420012 (1)c | ||
| 430012 (5)c | ||
| 430212 (1)c | ||
| 630212 (1)e | ||
| Not identifiable | NAf | 030012 (1) |
| 430002 (1) | ||
| 630006 (1) | ||
| 630002 (1) | ||
| 630012 (8) |
Correct identifications registered an excellent, very good, good, implicit, satisfactory, or adequate biocode.
Not-identifiable biocodes were the result of a probability overlap between two or more possibilities; additional tests were required or gave an incorrect or unidentified response.
Burkholderia cepacia.
CDC NO-1.
Stenotrophomonas maltophilia.
NA, not applicable.
The strains of B. pseudomallei and B. mallei that were correctly identified, misidentified, or not identified by either system were not associated by common geography, source, or time period.
Conclusion.
In this study, all test results were intentionally based upon a single test, and no additional testing was performed. Other studies reported retesting and/or supplementing these rapid tests with additional traditional biochemical tests (1, 3, 5). This preliminary evaluation did not find either of these systems in the current format to be promising for confirmatory identification of potential B. pseudomallei or B. mallei, and therefore, we did not pursue a further major validation study. In addition to the poor performance of both the API 20NE and RapID NF Plus systems, we encountered other problems while working with them. While the RapID NF Plus requires only a 4-h incubation, an extensive (48-h) incubation of the API test strips was required, which is a disadvantage in terms of rapid response (1, 3). However, as it did not correctly identify any B. pseudomallei isolate, the speed of the RapID NF Plus systems confers no real advantage over the API 20NE. Safety was also a concern. The potential aerosolization from the manipulation of suspensions, the open-reaction cupules on the test strips, and the sharp edges generated from snapping open glass tube API reagents present opportunities for laboratory-acquired infection or injury (5).
To be beneficial in the detection of B. pseudomallei and B. mallei, these systems need to expand their databases to include a wider diversity of strains and/or adjust problematic biochemical tests within the test panels. Consequently, we continue to recommend the use of traditional biochemical methods for preliminary identification of these agents, followed by submission of suspicious isolates to a laboratory capable of confirmatory identification (1).
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