Evaluation of the Duopath Legionella Lateral Flow Assay for Identification of Legionella pneumophila and Legionella Species Culture Isolates

Abstract

Duopath Legionella (Merck KGaA, Darmstadt, Germany) is a new immunochromatographic assay for the simultaneous identification of cultured L. pneumophila and Legionella species other than L. pneumophila. In tests of 89 L. pneumophila strains and 87 Legionella strains other than L. pneumophila representing 41 different species, Duopath and a widely used latex agglutination assay detected L. pneumophila with 100% and 98% accuracy, respectively, whereas the percentages differed significantly for other Legionella spp. (93% versus 37% [P < 0.001]). Since many countries’ regulations require the identification of Legionella spp. in water and environmental samples, the use of Duopath Legionella to comply with those regulations could contribute to significantly fewer false-negative results.

Legionellae are ubiquitous bacteria in the natural aquatic environment and often colonize man-made aquatic environments. When inhaled in aerosol form, legionellae may cause a severe, atypical pneumonia named Legionnaires' disease. Currently, the genus Legionella is known to include 50 species (see J. P. Euzéby's List of Prokaryotic Names with Standing in Nomenclature [www.bacterio.cict.fr/]). Some of the species have been isolated from only environmental sources to date, but it is generally accepted that all species may cause pneumonia, especially in immunocompromised persons. For monitoring of water systems, the “gold standard” method in a routine laboratory for identification of legionellae is enrichment on glycine-vancomycin-polymyxin B-cycloheximide (GVPC) agar plates with subsequent confirmation that is mostly done by serological methods. Unfortunately, the huge number of Legionella spp. and L. pneumophila serogroups represent a very wide serological heterogeneity which can lead to false-negative results. Molecular methods could circumvent this limitation, but these methods are generally not user-friendly and convenient. A new immunochromatographic identification (lateral flow) assay named Duopath Legionella has been recently developed by Merck KGaA and is intended for the simultaneous recognition of L. pneumophila and other Legionella spp. on the same test device. Separate recognition of L. pneumophila within the genus Legionella is based on the use of monoclonal antibodies that recognize species-specific and genus-specific epitopes of the Mip proteins (3). Here we describe the evaluation of this new assay in comparison with the widely used latex agglutination assay (Legionella latex test) from Oxoid, Basingstoke, United Kingdom, which recognizes the most important Legionella spp. causing pneumonia. While the latex assay contains latex suspensions for other Legionella spp. separate from those for L. pneumophila serogroup 1 and for serogroups 2 to 14, Duopath Legionella is composed like a pregnancy test, with separate detection zones for Legionella spp. and for L. pneumophila (all serogroups) on the same test device.

For evaluation of Duopath Legionella, Legionella type strains or water samples were cultured on GVPC agar (Merck, Darmstadt, Germany) for 3 to 5 days. The prolongation of growth for up to 2 weeks had no influence on the results (data not shown). Patient isolates were grown on BMPA agar (Oxoid, Wesel, Germany). One suspect Legionella colony (approximate colony diameter, 1 to 2 mm) was resuspended in a 0.9% NaCl solution containing 1% Tween 20. After the addition of polymyxin B (Bacillus cereus selective supplement; Merck, Darmstadt, Germany), the suspension was incubated for 2 to 5 min at room temperature followed by 5 min at 95°C and cooled to room temperature for pipetting into the sample port of Duopath Legionella. Results at test and control zones were read after 30 min without a magnifying glass. The ability of Duopath Legionella to identify L. pneumophila and other Legionella spp. was compared with that of the appropriate latex assay of Oxoid recognizing L. pneumophila serogroup 1, L. pneumophila serogroups 2 to 14, or other Legionella spp. Tests were performed according to the manufacturer's description.

Specificity of Duopath Legionella.

The specificity was calculated by testing a total of 87 bacterial strains isolated from water samples and grown on GVPC agar plates (Merck, Darmstadt, Germany) or from human sources and grown on BMPA agar (Oxoid, Wesel, Germany). All of these strains were able to grow on blood agar (Merck, Darmstadt, Germany), which is the first exclusion criterion for identification of Legionella spp. Afterwards they were identified using the API system (bioMerieux, Nürtingen, Germany) as Acinetobacter baumannii (2 strains), Acinetobacter haemolyticus (2), Acinetobacter sp. (3), Aeromonas hydrophila (1), Alcaligenes faecalis (1), Brevundimonas diminuta (1), Burkholderia cepacia (4), Citrobacter freundii (2), Escherichia coli (2), Pseudomonas aeruginosa (40), Pseudomonas alcaligenes (2), Pseudomonas putida (2), Pseudomonas stutzeri (1), Pseudomonas sp. (4), Serratia marcescens (2), and Stenotrophomonas maltophilia (18). None of those bacteria tested positive by Duopath Legionella; thus, the specificity of the testing was 100%.

Identification of L. pneumophila.

A total of 89 L. pneumophila strains were tested with both assays. The details of the strains and isolates tested were as follows: (i) type strains of monoclonal subgroups of serogroup 1 (n = 10) according to Joly et al. (4), (ii) ATCC serogroup type strains of serogroups 2 to 15 (n = 14), and (iii) water or patient isolates (n = 65) confirmed as L. pneumophila by use of MONOFLUO anti-Legionella staining reagent (Bio-Rad, Munich, Germany). By Duopath Legionella, all of them were detected as providing bands at the detection zones for both L. pneumophila and other Legionella spp. When the agglutination assays for serogroup 1 and serogroups 2 to 14 were used, two of the L. pneumophila isolates gave reproducibly negative results with the Oxoid test. The possibility of a prozone phenomenon was excluded. Serogroup typing of these strains failed with rabbit sera and serogroup-specific monoclonal antibodies produced in our laboratory (2) as well as with monovalent fluorescein isothiocyanate-conjugated anti-Legionella (serogroups 1 to 14) rabbit sera (Prolab Diagnostics, Neston, United Kingdom).

Recognition of Legionella spp.

For recognition of Legionella spp., 42 Legionella ATCC or NCTC type strains belonging to 39 different species other than L. pneumophila were included in this study (Table 1). For three species (L. bozemanae, L. longbeachae, and L. spiritensis), members of both known serogroups were analyzed. In addition, 45 environmental isolates belonging to 20 Legionella spp. (one to five strains per species) were tested. These isolates were classified on the species level by mip gene sequencing (5) according to the guidelines and databases of the European Working Group for Legionella Infections, which are available in the web site www.ewgli.org (link: “Typing and Identification Schemes”).

TABLE 1.

Identification of strains of Legionella other than L. pneumophila by Duopath Legionella and latex agglutination assay

Legionella sp. (serogroup)	Designation	Legionella type strain test resulta		No. of water isolates positive/ total no. testedb
		Duopath Legionella	Latex agglutination	Duopath Legionella	Latex agglutination
L. adelaidensis	ATCC 49625	o	o	0/0	0/0
L. anisa	ATCC 35292	+	+	2/2	2/2
L. birminghamensis	ATCC 43702	+	o	0/0	0/0
L. bozemanae (1)	ATCC 33217	+	+	0/0	0/0
L. bozemanae (2)	ATCC 35545	+	+	0/0	0/0
L. bozemanae				3/4	4/4
L. brunensis	ATCC 43878	+	o	0/0	0/0
L. cherrii	NCTC 11976	+	+	1/1	1/1
L. cincinnatiensis	NCTC 12438	+	+	0/0	0/0
L. dumoffii	ATCC 33279	+	+	1/1	1/1
L. erythra (1)	ATCC 35303	+	o	1/1	0/1
L. fairfieldensis	ATCC 49588	+	o	0/0	0/0
L. feeleii (1)	ATCC 35072	+	o	0/0	0/0
L. feeleii				4/4	0/4
L. geestiana	ATCC 49504	+	o	0/0	0/0
L. gormanii	ATCC 33297	+	+	1/1	1/1
L. gratiana	ATCC 49413	+	+	0/0	0/0
L. hackeliae (1)	ATCC 35250	+	o	0/0	0/0
L. israelensis	NCTC 12010	+	o	0/0	0/0
L. jamestowniensis	ATCC 35298	+	o	1/1	0/1
L. jordanis	ATCC 33623	+	+	2/2	2/2
L. lansingensis	ATCC 49751	+	o	0/0	0/0
L. londiniensis	ATCC 49505	+	o	3/3	0/3
L. longbeachae (1)	ATCC 33462	+	+	0/0	0/0
L. longbeachae (2)	ATCC 33484	+	+	0/0	0/0
L. maceachernii	ATCC 35300	+	o	5/5	0/5
L. micdadei	ATCC 33218	+	+	3/3	3/3
L. moravica	ATCC 43877	+	o	0/0	0/0
L. nautarum	ATCC 49506	+	o	0/0	0/0
L. oakridgensis	ATCC 33761	+	o	1/1	0/1
L. parisiensis	ATCC 35299	+	+	0/0	0/0
L. quateirensis	NCTC 12376	+	o	0/0	0/0
L. quinlivanii (1)	ATCC 43830	+	o	0/0	0/0
L. rubrilucens	ATCC 35304	+	o	4/4	0/4
L. sainthelensi (1)	ATCC 35248	+	o	1/1	0/1
L. santicrucis	NCTC 11989	o	o	0/0	0/0
L. shakespearei	ATCC 49655	+	o	0/0	0/0
L. spiritensis (1)	NCTC 11990	+	o	0/0	0/0
L. spiritensis (2)	NCTC 12082	+	o	0/0	0/0
L. steigerwaltii	ATCC 35302	o	+	3/3	3/3
L. taurinensis				5/5	0/5
L. tucsonensis	NCTC 12439	+	+	1/1	0/1
L. wadsworthii	ATCC 33877	o	o	0/0	0/0
L. waltersii	NCTC 13017	+	o	0/0	0/0
L. worsleiensis	ATCC 49507	+	o	1/1	0/1
Legionella genomospecies A				1/1	0/1

^a+, positive; o, negative. Of 42 strains tested, 38 (90.5%) gave positive results by Duopath Legionella and 15 (35.7%) gave positive results by the latex agglutination assay.

^bOf 45 strains tested, 43 (95.6%) gave positive results by Duopath Legionella and 17 (37.8%) gave positive results by the latex agglutination assay.

Duopath Legionella recognized 38 (90%) of the 42 Legionella type strains other than L. pneumophila (Table 1), while the latex assay recognized only 15 of these 42 strains (36%). In tests of the environmental isolates, Duopath Legionella was positive for 43 of the 45 strains tested (96%) and the latex assay was positive for 17 (38%). Interestingly, for Duopath Legionella there was no absolute agreement to the species level represented by the type strains as seen for L. steigerwaltii. The three environmental isolates’ test results were positive, whereas the type strain result was negative. With all positive-testing Legionella strains other than L. pneumophila, Duopath Legionella always provided a specific signal only at the Legionella sp. test zone but never at the L. pneumophila test zone.

Superiority of Duopath Legionella over the latex assay for identification of legionellae in water systems.

In summary, the latex agglutination assay is aimed at recognizing the Legionella species most frequently causing Legionnaires' disease (L. anisa, L. bozemanae, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, and L. micdadei) but not the wide range of legionellae found in water systems, which are also suspected to be pneumonia pathogens. According the manufacturer's instructions, cross-reactions have been reported to occur occasionally with certain serotypes with at least eight other Legionella spp. Among all of the Legionella strains other than L. pneumophila involved in this study (n = 87), the Duopath test correctly detected 93% whereas the agglutination assay identified significantly fewer (37% [P < 0.001]).

The guidelines of the Centers for Disease Control and Prevention (1) and of the European Working Group for Legionella Infections (www.ewgli.org [link: “EQA Water Scheme”]) for monitoring of water systems recommend testing for other Legionella spp. as well as for L. pneumophila. Given the low number of Legionella spp. recognized in our testing, it can be assumed that a significant number of false-negative results occur when the latex assay is used. Here, Duopath Legionella revealed that it possesses an important advantage over the latex assay and that its use would make the phenotypic diagnostic gap significantly smaller. Therefore, Duopath Legionella can be considered a user-friendly, simple, and reliable test for the simultaneous identification of L. pneumophila and other Legionella strains.