Jpn. J. Infect. Dis., 60, 214-216, 2007

Short Communication Comparative Evaluation of Duopath Lateral Flow Assay against the Conventional Culture Method Using and Legionella anisa Strains Michio Koide*, Shusaku Haranaga, Futoshi Higa, Masao Tateyama, Nobuhisa Yamane1 and Jiro Fujita Department of Medicine and Therapeutics, Control and Prevention of Infectious Diseases (First Department of Internal Medicine) and 1Department of Clinical Laboratories, Faculty of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan (Received January 26, 2007. Accepted April 5, 2007) SUMMARY: Duopath Legionella is a new immunochromatographic assay for the identification of Legionella pneumophila and Legionella spp. As excellent specificity has been previously reported for this kit, we attempted an evaluation of its sensitivity using L. pneumophila serogroup 1 and Legionella anisa strains. Bacterial suspen- sions of L. pneumophila at concentrations of 1.2×108 cfu/ml and 1.2×107 cfu/ml were detected, but those below 1.2×106 cfu/ml were not recognized by the Duopath kit. After centrifugation and the sediment resuspension, a 2.8×107 cfu/ml concentration of bacterial suspension showed a positive result, but negative results were obtained below 2.8×106 cfu/ml. Also, bacterial suspension with concentrations of 3.2×109 cfu/ml and 1.4×109 cfu/ml after centrifugation of L. anisa were detected, but those below 3.2 × 108 cfu/ml and 1.4 × 108 cfu/ml after centrifugation were not recognized by the Duopath kit. Meanwhile, this kit was less sensitive to the L. pneumophila serogroup 1 suspension, and was more sensitive to the L. pneumophila serogroup 2 and L. anisa than the Binax NOW immunochromatographic kit. It was realized that the sensitivity of this kit is too low for determining the presence of Legionella in water samples. Although this kit may have excellent specificity, it has low sensitivity.

Legionella, the causative agent of Legionnaires’ disease, is the serial 10-fold dilutions (10–4, 10–5, 10–6, 10–7) was added a type of that is ubiquitous in natural and man-made to each 200-ml sampling bottle containing 200 ml of sterile aquatic environments. The Duopath Legionella (Merck KGaA, tap water. Each sample was centrifuged at 8,000 g for 20 Darmstadt, Germany) is a new immunochromatographic as- min. The resultant sediment was resuspended in 1 ml of say for the simultaneous identification of cultured Legionella supernatant so the number of Legionella colony units formed pneumophila and Legionella spp. other than L. pneumophila could be counted. We then injected 1 ml of 0.2M HCl-KCl (1). In performing microbiological analysis of water samples buffer (pH 2.2) into this suspension and allowed the mixture in order to prevent Legionnaires’ disease, testing should be to stand at room temperature for 30 min. A one hundred done not only for the presence of L. pneumophila, but also microliter portion of this buffer-treated suspension was plated for the genus Legionella itself. The manufacturer’s instruc- on a BCYEα agar medium. The preparations were incubated tions also suggested that, in addition to conventional culture at 35°C for 7 days (2). methods, users prefer immunological techniques due to their For the evaluation of Duopath Legionella, 1 ml or 10 ml higher sensitivity and specificity. As the excellent specificity amounts from the original Legionella suspensions and from of this kit has been previously reported, we attempted an the suspensions that had been centrifuged were centrifuged evaluation of its sensitivity (1). at 12,000 g for 30 min. The sediment was resuspended in 144 For this comparison of a conventional culture method and μl of 0.9% NaCl solution containing 1% Tween 20. After the Duopath kit, the L. pneumophila serogroup 1 type strain the addition of polymyxin B (Bacillus cereus selective supple- Philadelphia-1 (ATCC33152) and the L. anisa SR strain, ment; Merck), the suspension was incubated for 5 min at room which was isolated from shower water collected at the Ryukyu temperature followed by 5 min at 95°C and subsequently University Hospital in 2005, were used (2). The Legionella cooled to room temperature for pipetting into the sample port strains were grown on buffered charcoal yeast extract agar of the Duopath Legionella. The results were read at the test supplemented with 0.1% α-ketoglutarate (BCYEα; Oxoid, and control zones after 30 min without using a magnifying Hampshire, UK) at 35°C for 3 days, and suspended with sterile glass. distilled water at an approximate concentration of 108 cfu/ First, suspensions were made of the L. pneumophila Phila- ml. Serial 10-fold dilutions were then performed. delphia-1 strain at a concentration of 1.2× 108 cfu/ml. The For the conventional culture method, 1 ml from each of original and serial 10-fold dilutions (10–1, 10–2, 10–3) of this bacterial suspension were examined using the Duopath kit. Bacterial suspensions of 1.2×108 and 1.2×107 cfu/ml were *Corresponding author: Mailing address: Department of Medi- cine and Therapeutics, Control and Prevention of Infectious detected from bands provided at a detection zone for L. Diseases (First Department of Internal Medicine), Faculty of pneumophila but not at a detection zone for other Legionella 6 Medicine, University of the Ryukyus, 207 Uehara, Nishihara- spp. Bacterial suspensions below 1.2 × 10 cfu/ml were not cho, Okinawa 903-0215, Japan. Tel: +81-98-895-1144, Fax: +81- recognized by the Duopath kit. One mililiter from each of 98-895-1414, E-mail: [email protected] these serial bacterial suspensions was added to each 200-ml

214 Table 1. Comparison of conventional culture method and Duopath immunochromatographic assay using Legionella pneumophila serogroup 1 and Legionella anisa strains Strains used at Culture Duopath kit After Culture Duopath kit original suspension cfu/ml L. pneumophila zone Legionella spp. zone centrifugation cfu/ml L. pneumophila zone Legionella spp. zone

L. pneumophila 1.2 ×108 + – L. pneumophila 2.8 ×107 + – original suspension 1.2 ×107 + – after centrifugation 2.8 × 106 –– 1.2 ×106 –– 2.8 ×105 –– 1.2 ×105 –– 2.8 ×104 –– L. anisa 3.2 ×109 – + L. anisa 1.4 ×109 – + original suspension 3.2 ×108 ––after centrifugation 1.4 ×108 –– 3.2 ×107 –– 1.4 ×107 –– 3.2 ×106 –– 1.4 ×106 –– +, positive; –, negative.

Table 2. Comparison of Binax NOW Urinary Antigen Test and Duopath immunochromatographic assay using Legionella pneumophila and Legionella anisa strains Legionella counts Duopath kit Strains used Binax NOW kit cfu/ml L. pneumophila zone Legionella spp. zone

L. pneumophila 2.6 ×108 + – + serogroup 1 2.6 ×107 + – + type strain 2.6 ×106 –– + 2.6 ×105 –– + 2.6 ×104 –– + 2.6 ×103 –– – 2.6 ×102 –– – L. pneumophila 3.6 ×109 ++ – serogroup 2 3.6 ×108 ++ – clinical isolate 3.6 ×107 + –– 3.6 ×106 –– – L. anisa 7.1 ×109 – + – environmental isolate 7.1 ×108 – + – 7.1 ×107 –– – 7.1 ×106 –– – +, positive; –, negative. sampling bottle containing 200 ml of sterile tap water. After L. pneumophila, we could not compare them (3). So we centrifugation and polymyxin B treatment, a 2.8×107 cfu/ml compared the sensitivity of these two kits by using the L. concentration of the bacterial suspension showed a positive pneumophila serogroup 2 OK strain (an isolate of this patient result for the L. pneumophila detection zone. The suspensions in Okinawa, 2007), in addition to the L. pneumophila showed negative results below 2.8 × 106 cfu/ml (Table 1). serogroup 1 Philadelphia-1 and the L. anisa SR strains. Next, suspensions were made of the L. anisa SR strain at a These strains were grown on BCYEα agar and treated concentration of 3.2×108 cfu/ml. The 10-fold concentration as described above (Tween 20-polymyxin B). Suspensions of of this suspension was detected from bands provided at a L. pneumophila serogroup 1 with concentrations greater than detection zone for Legionella spp., but not at a detection zone 2.6 × 107 cfu/ml were recognized by the Duopath kit at a for L. pneumophila. Bacterial suspensions below 3.2 × 108 detection zone for L. pneumophila, and those with concentra- cfu/ml were not recognized by the Duopath kit. Ten milliliters tions greater than 2.6×104 were detected by the Binax NOW and 1 ml amounts from the original suspensions and 1 ml kit (Table 2) (4). The Binax NOW kit was a thousand times from each of the serial 10-fold dilutions (10–1, 10–2) were more sensitive than the Duopath kit. Conversely, in the case added to each 200-ml sampling bottle. After centrifugation of the L. pneumophila serogroup 2 suspension, the Duopath and polymyxin B treatment, the bacterial suspension with the kit detected a 3.6 × 107 bacterial suspension at a detection 1.4× 109 cfu/ml concentration showed a positive result for zone for L. pneumophila and detected suspensions greater Legionella spp. in the detection zone. Suspensions showed than 3.6 × 108 at detection zones for both L. pneumophila negative results below 1.4 × 108 cfu/ml (Table 1). and other Legionella spp., but the Binax NOW kit showed On the other hand, the Duopath kit showed negative re- negative results with concentrations as high as 3.6 × 109. sults for the urine of legionellosis patients that had showed The Duopath kit showed almost the same sensitivity to L. positive results with the Binax NOW immunochromatographic pneumophila serogroup 2 as to L. pneumophila serogroup 1. kit (Binax, Portland, Maine, USA). The urine of the patient In the case of L. anisa suspension, the Duopath kit detected a whose illness was caused by L. pneumophila serogroup 2 7.1 × 108 bacterial suspension at a detection zone for other showed negative results with both the Duopath and Binax Legionella spp., but the Binax NOW kit showed negative NOW kits. Regrettably, as we did not store urine samples results with concentrations as high as 7.1 × 109. The Binax of legionellosis patients infected by bacteria other than NOW kit had an excellent sensitivity specific for the L.

215 pneumophila serogroup 1 only. Duopath lateral flow assay for identification of Legionella pneumophila Consequently, the sensitivity of the Duopath Legionella and Legionella culture isolates. Appl. Environ. Microbiol., 72, 7 4489-4491. Lateral Flow Assay was found to be 10 cfu for L. pneumophila 2. Koide, M., Owan, T., Nakasone, C., et al. (2007): Prospective monitor- and 109 cfu for L. anisa. It was realized that the sensitivity of ing study: isolating Legionella pneumophila in a hospital water system this kit is too low to determine the presence of Legionella in located in the obstetrics and gynecology ward after eradication of water samples. If we were to use this kit for the direct detec- Legionella anisa and reconstruction of shower units. Jpn. J. Infect. Dis., 60, 5-9. tion of aquatic samples, it is possible that most samples would 3. Koide, M., Higa, F., Tateyama, M., et al. (2006): Detection of Legionella be judged to be negative despite containing high concentra- species in clinical samples: comparison of polymerase chain reaction tions of Legionella, in some cases as high as 106 cfu/ml. and urinary antigen detection kits. Infection, 34, 264-268. Although this kit may have excellent specificity, it has low 4. Helbig, J.H., Uldum, S.A., Lück, P.C., et al. (2001): Detection of sensitivity. Legionella pneumophila antigen in urine samples by the Binax NOW immunochromatographic assay and comparison with both Binax Legionella Urinary Enzyme Immunoassay (EIA) and Biotest Legionella REFERENCES Urine Antigen EIA. J. Med. Microbiol., 50, 509-516. 1. Helbig, J.H., Lück, P.C., Kunz, B., et al. (2006): Evaluation of the

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