Multicenter Comparison of Molecular Methods for Detection of
            <i>Legionella</i>
            spp. in Sputum Samples

Bencini, Max A.; Brule, Adriaan J. C. van den; Claas, Eric C. J.; Hermans, Mirjam H. A.; Melchers, Willem J. G.; Noordhoek, G. T.; Salimans, M. M. M.; Schirm, J; Vink, Cornelis; Zee, Anneke van der; Jansen, Ruud

doi:10.1128/jcm.00505-07

Cited by 18 publications

(10 citation statements)

References 14 publications

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“…Previously, mucolytic agents, haemoglobin and other undefined substances in sputum samples have been reported to inhibit PCR (Wilson, 1997;Amicosante et al, 1995, Deneer & Knight, 1994. Pretreatment to reduce viscosity in respiratory samples prevents inhibition of PCR (Bencini et al, 2007). Overall, the specimen type and use of an internal control for the detection of inhibition are the two major aspects that influence the reliability of diagnostic P. jirovecii PCR methods.…”

Section: Discussionmentioning

confidence: 99%

Degree and frequency of inhibition in a routine real-time PCR detecting Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia in Turkey

Döşkaya

Caner

Değirmenci

et al. 2011

Journal of Medical Microbiology

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Routine laboratory diagnosis of Pneumocystis jirovecii is currently achieved by PCR in almost all laboratories with sufficient equipment due to its high sensitivity and specificity compared to staining methods. A current issue that limits the reliability and sensitivity of PCR is the degree of inhibition caused by inhibitory substances in respiratory samples. The present study aimed to analyse the degree and frequency of inhibition in real-time PCR detecting P. jirovecii in respiratory specimens submitted to a Pneumocystis pneumonia (PcP) diagnosis laboratory in Ege University Medical School, Turkey. Between July 2009 and December 2010, 76 respiratory specimens [63 bronchoalveolar lavage (BAL) fluid, 10 sputum samples, two tracheal aspiration fluid and one thoracentesis fluid] obtained from 69 PcP-suspected patients were investigated for the presence of P. jirovecii using real-time PCR targeting the cdc2 gene. Of these samples, 42 of the specimens were stained and examined by microscopy according to the request of the clinicians. PCR was positive in 15 specimens in the initial run. Of the remaining 61 samples, 41 of them were negative with positive internal inhibition controls (i.e. true-negative group). The frequency of inhibition in the initial run was 26.31 % (20/76) as determined by spiked negative controls. All of the inhibited samples were resolved after 1 : 2, 1 : 5, 1 : 10 and 1 : 20 dilutions. P. jirovecii was detected by PCR in two inhibited specimens after retesting with diluted samples which were also positive by microscopy. The incidence of P. jirovecii in respiratory specimens was 22.36 % (17/76) as determined by real-time PCR and 7.14 % (3/42) by microscopy. Overall, the incidence of P. jirovecii in respiratory samples was 23.68 % (18/76) as detected by both methods. In conclusion, inclusion of spiked positive controls in each sample and retesting with diluted samples to resolve inhibition increased the reliability of the real-time PCR assay in terms of determining false-negative results and influencing the treatment of the patient. Furthermore, results of the present study determined for the first time the frequency and degree of inhibition in a real-time PCR detecting P. jirovecii in respiratory specimens during routine diagnosis of PcP.

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Section: Discussionmentioning

confidence: 99%

Degree and frequency of inhibition in a routine real-time PCR detecting Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia in Turkey

Döşkaya

Caner

Değirmenci

et al. 2011

Journal of Medical Microbiology

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“…Commercial and inhouse-developed NAATs are used in both clinical and environmental laboratories; however, the list of Legionella genes amplified is limited. The most common targets include a conserved segment of the rRNA genes for the 5S and 16S subunits, the 16S-23S spacer, and/or the macrophage inhibitor protein mip, found primarily in the genus Legionella and highly conserved in all L. pneumophila isolates (226,(332)(333)(334)(349)(350)(351)(352)(353)(354)(355)(356)(357)(358). Several studies have also examined alternative chromosomal targets, such as dotA, gyrB, dnaJ, wzm, and wzt (340).…”

Section: Nucleic Acid-based Molecular Diagnosticsmentioning

confidence: 99%

Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations

2015

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SUMMARY Legionnaires' disease (LD) is an often severe and potentially fatal form of bacterial pneumonia caused by an extensive list of Legionella species. These ubiquitous freshwater and soil inhabitants cause human respiratory disease when amplified in man-made water or cooling systems and their aerosols expose a susceptible population. Treatment of sporadic cases and rapid control of LD outbreaks benefit from swift diagnosis in concert with discriminatory bacterial typing for immediate epidemiological responses. Traditional culture and serology were instrumental in describing disease incidence early in its history; currently, diagnosis of LD relies almost solely on the urinary antigen test, which captures only the dominant species and serogroup, Legionella pneumophila serogroup 1 (Lp1). This has created a diagnostic “blind spot” for LD caused by non-Lp1 strains. This review focuses on historic, current, and emerging technologies that hold promise for increasing LD diagnostic efficiency and detection rates as part of a coherent testing regimen. The importance of cooperation between epidemiologists and laboratorians for a rapid outbreak response is also illustrated in field investigations conducted by the CDC with state and local authorities. Finally, challenges facing health care professionals, building managers, and the public health community in combating LD are highlighted, and potential solutions are discussed.

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“…The detection of other species besides L. pneumophila is relevant to the diagnosis of Legionnaires' disease, since this disease can be caused by other species, such as L. longbeachae and L. bozemanii. The correct identification of species is also important for epidemiological studies and identification of sources of infection [65]. However, the major disadvantage of PCR it is the inability to evaluate the viability, in other words, by the PCR technique it is not possible to distinguish between viable and nonviable microorganisms, detecting only their presence or absence, while only viable bacteria are able to cause infections in human and represent an interest for public health [20,23,47].…”

Section: Discussionmentioning

confidence: 99%

Detection of <i>Legionella</i> spp. in Natural and Man-made Water Systems Using Standard Guidelines

Borges¹,

Simes²,

Martínez‐Murcia³

et al. 2012

MICROBIOLOGY

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Infections caused by Legionella spp. are considered at the present time, an emerging public health problem and are linked to high rates of mortality and morbidity, if not properly treated. In this study were analyzed 54 samples of water from 8 counties at Northern Portugal, with the aim of obtaining a collection of strains of the genus Legionella and to characterize them genetically and phenotypically. Another objective of this study was to evaluate the effectiveness of the technique of cultivation, a standard method according to International Organization for Standardization ISO 11731:1998, for detection and enumeration of species of Legionella. For laboratory processing, after the filtration of samples (1 L), the filtrate was resuspended in sterile distilled water (5 ml). Heat treatment for selective inhibition of non-Legionella bacteria was performed. Subsequently, 100 μl of the suspension was spread in GVPC selective agar medium, and incubated (7 to 10 days) at 37 ℃. Colonies that were morphologically characteristic of the genus were sub-cultured onto BCYE agar and blood agar for verification. According to the procedure recommended by the standard method, only the colonies which grew in BCYE agar and not on blood agar were considered as suspected Legionella strains. The identification of these initially selected colonies was performed by sequencing the 16S rRNA gene, which revealed that none of the isolates were identified as belonging to the genus Legionella. However, through the ISO 11731:1998 they were interpreted as positive, corresponding therefore to "false-positive" results. The methods used in this study allowed the isolation of a number of isolates (40), which form an independent group of all genus of the family Chitinophagaceae outlined so far, and that by their phylogenetic distance might be a genus not yet described and therefore a new species. The results obtained, highlighted the importance of using culture and genetic methods in parallel for the proper identification of microorganisms.

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Multicenter Comparison of Molecular Methods for Detection of Legionella spp. in Sputum Samples

Cited by 18 publications

References 14 publications

Degree and frequency of inhibition in a routine real-time PCR detecting Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia in Turkey

Degree and frequency of inhibition in a routine real-time PCR detecting Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia in Turkey

Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations

Detection of <i>Legionella</i> spp. in Natural and Man-made Water Systems Using Standard Guidelines

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