Hospital acquired pneumonia: Comparison of culture and real-time PCR assays for detection of Legionella pneumophila from respiratory specimens at Tehran Hospitals

Abstract

Legionella pneumophila is an important etiological agent in both hospital and community acquired pneumonia. The sensitivity of culture for isolation of L. pneumophila from clinical specimens is low and time consuming. Similar problem also exists when the method of direct immunofluorescence is used. To detect this organism quantitatively from respiratory specimens, a Taq Man based real-time PCR targeting the mip sequence was developed. Both real-time PCR and culture methods were applied on 262 respiratory specimens from 262 ICU patients with pneumonia admitted to 5 different hospitals in Tehran. The results of real-time PCR were compared with those obtained by culture. Real-time PCR and culture found 12 and 4 specimens, respectively, as positive for L. pneumophila. Its technical specificity (100%) was checked against a panel of microorganisms consisting of both Gram-positive and Gram-negative bacteria. Our real-time PCR assay showed high sensitivity (100%) and specificity (96.9%) and could detect 200 organisms per ml from respiratory specimens. Using real-time PCR as a screening method, the frequency of nosocomial pneumonia with L. pneumophila at Tehran hospitals was estimated as 4.58%.

Comparison of in situ sequence type analysis of Legionella pneumophila in respiratory tract secretions and environmental samples of a hospital in East Jerusalem

“…On the other hand, molecular diagnostic techniques for L. pneumophila from respiratory samples, such as polymerase chain reaction (PCR), are well established and increasingly adopted worldwide [2,11]. Endpoint and real-time PCR offer substantial advantages over cultivation for the detection and quantification of L. pneumophila, including high sensitivity, specificity and time efficiency [12][13][14][15]. For epidemiological studies, in addition to detection and diagnosis, several rapid and discriminatory methods have been developed for Legionella typing; DNA banding pattern-based and DNA sequencing-based genotyping (among others, Ref) are widely used methods for subtyping of bacteria.…”

Prevalence of Legionella pneumophila among patients suffering from pneumonia: a Middle East systematic review and meta-analysis

“…In general, 58.8 and 41.2% of patients were male and female, respectively (Table 1). Phenotyping methods included culture on BCYE medium [7,[12][13][14][15][16][17], serological tests (direct immunofluorescence [12], determination of IgM titer [ELISA] kit [13], IgM and IgG (EIA kits) [15,18], urine antigenic test [UAT]) [8] and molecular techniques: nested PCR sequence-based typing (NPSBT) [16], PCR of mip gene [17,19] and LAMP [19], RAPD-PCR [20], real-time PCR of 16s rRNA gene [7,8].…”

A Review of Approaches to Identify Enteric Bacterial Pathogens

“…Real-time PCR combines PCR chemistry with fluorescent probe detection of the amplified product. This method is simpler to carry out compared to conventional PCR method and its test result comes much sooner too ( 39 , 40 ). Two kinds of chemical agents are available for real-time PCR products: fluorescent probes that bind specifically to definite DNA sequences and fluorescent dyes that intercalate into any dsDNA ( 41 ).…”