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Applied and Environmental Microbiology, October 2006, p. 6859, Vol. 72, No. 10
0099-2240/06/$08.00+0     doi:10.1128/AEM.00968-06

LETTER TO THE EDITOR

Caution on Interpretation of Legionella Results Obtained Using Real-Time PCR for Environmental Water Samples

Top Letter References

 
In an article entitled "Quantitative Real-Time Legionella PCR for Environmental Water Samples: Data Interpretation" (2), the authors reported their results using quantitative real-time PCR to detect Legionella in environmental water samples. The authors found high false-positive rates for the PCR test if culture was used as the reference standard (Table 1), which is in agreement with the work of other investigators (3, 5) who found a higher positive rate of water samples with the PCR method than with culture, and the authors suggested that the high positive rate may be due to the presence of viable but nonculturable Legionella cells in water samples (6). However, no data were provided.

View this table: [in this window] [in a new window]

TABLE 1. Sample positivity comparison by culture and PCR methodsa

PCR detects both viable and nonviable cells by amplifying the target nucleic acids in the sample based on the assumption that the nucleic acids being copied are from live cells (4). We wish to postulate another hypothesis: the false-positive results are due to the presence of nonviable Legionella cells in water samples. Disinfection of cooling towers and potable water systems for Legionella have been applied widely. If disinfection is performed, it is likely that the water samples will contain nonviable Legionella cells which were killed by the disinfection measures. The remaining nucleic acids in the dead cells may still be recovered and amplified by the PCR. This can explain why no correlation was observed between culture and PCR results.

The detection of viable Legionella cells may be achieved by the detection of mip mRNA as the target, reverse transcription (to form cDNA), and then PCR amplification. The basis of detecting mRNA and not rRNA is that most of the bacterial mRNA have a short half-life (<2 min). Thus, detecting mip mRNA would indicate the presence of metabolically active (living) Legionella cells that must be present within a few minutes prior to the sample process. However, this method is technically more difficult and less sensitive (1).

False-positive readings of Legionella samples could lead to unnecessary and expensive emergency decontamination procedures. Using the PCR result for Legionella samples may overestimate the risk of infection. The PCR results must be applied with caution. Culture remains the reference standard for testing Legionella in environmental samples.

 
Ed. Note: The authors of the published article declined to respond.

Top Letter References

 

1
1. Eej, A. K., M. H. Mahbubani, and R. M. Atlas. 1991. Detection of viable Legionella pneumophila in water by polymerase chain reaction and gene probe methods. Appl. Environ. Microbiol. 57:597-600.[Abstract/Free Full Text]
2
2. Joly, P., P.-A. Falconnet, J. André, N. Weill, M. Reyrolle, F. Vandenesch, M. Maurin, J. Etienne, and S. Jarraud. 2006. Quantitative real-time Legionella PCR for environmental water samples: data interpretation. Appl. Environ. Microbiol. 72:2801-2808.[Abstract/Free Full Text]
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3. Wellinghausen, N., C. Frost, and R. Marre. 2001. Detection of legionellae in hospital water samples by quantitative real-time LightCycler PCR. Appl. Environ. Microbiol. 67:3985-3993.[Abstract/Free Full Text]
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4. Wilson, I. G. 1997. Inhibition and facilitation of nucleic acid amplification. Appl. Environ. Microbiol. 63:3741-3751.[Medline]
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5. Yamamoto, H., Y. Hashimoto, and T. Ezaki. 1993. Comparison of detection methods for Legionella species in environmental water by colony isolation, fluorescent antibody staining, and polymerase chain reaction. Microbiol. Immunol. 37:617-622.[Medline]
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6. Yamamoto, H., Y. Hashimoto, and T. Ezaki. 1996. Study of nonculturable Legionella pneumophila cells during multiple-nutrient starvation. FEMS Microbiol. Ecol. 20:149-154.[CrossRef]

						Hsiu-Yun Shih Yusen Eason Lin* Graduate Institute of Environmental Education National Kaohsiung Normal University 62 Shen-chong Rd. Yanchao Kaohsiung 824, Taiwan
						* Phone: 886-7-6051036, Fax: 886-7-6051151, E-mail: easonlin{at}nknucc.nknu.edu.tw

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Applied and Environmental Microbiology, October 2006, p. 6859, Vol. 72, No. 10
0099-2240/06/$08.00+0     doi:10.1128/AEM.00968-06

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Shih, H.-Y. Articles by Lin, Y. E. Search for Related Content PubMed PubMed Citation Articles by Shih, H.-Y. Articles by Lin, Y. E. Agricola Articles by Shih, H.-Y. Articles by Lin, Y. E.