Real-Time PCR Detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila in Respiratory Specimens Using the ARIES® System

Marimuthu, Subathra; Wolf, Leslie A.; Summersgill, James T.

doi:10.18297/jri/vol2/iss1/3/

Cited by 3 publications

(3 citation statements)

References 5 publications

(7 reference statements)

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“…Legionella pneumophila IgM detection by ELISA presented a low sensitivity (30%) especially with old age as IgM response is affected by immune status (Marimuthu et al,2018). According to multiplex-PCR performed, we classified the studied group into 2 groups; the PCR positive L. pneumophila group (12 patients) and the PCR negative L. pneumophila group (62 patients) and each group was related to clinical, laboratory and radiological parameters of the patients.…”

Section: Discussionmentioning

confidence: 99%

Molecular Detection of Bacterial Agents of Atypical Pneumonia: Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella pneumophila in Suez Canal region

El-Sayed

2021

Medicine Updates

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Background: Atypical bacterial infections played an important role in community-acquired pneumonia (CAP). It is difficult to detect atypical pathogens by conventional microbiological diagnostic methods. Atypical bacteria do not respond to beta-lactam antibiotics. The use of multiplex polymerase chain reaction (PCR) methods enables rapid and simultaneous detection of many pathogens in a single analysis. Aim: detecting the prevalence of atypical bacterial pathogens as etiologic agents of atypical pneumonia, in the Suez Canal region.Materials and Methods: This cross-sectional descriptive study was conducted throughout 18 months, from October 2018 to April 2020. It included 84 Egyptians suffered from CA atypical pneumonic patients of all age groups from Suez-Canal region, Egypt. Sputum samples were collected for identification of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella pneumophilia by using multiplex PCR.Results: Among the 84 atypical pneumonia patients, L. pneumophila were detected in 12 (14%) patients. M. pneumoniae and C. pneumoniae were not detected in our samples. Compared with L. pneumophila -negative cases, L. pneumophila -positive cases were more prevalent in middle aged males, smokers, COPD, diabetic and asthmatic patients (P values = 0.048). Persistent cough, elevated levels of C-reactive protein (C-RP), bilateral pulmonary infiltration are significant clues for predicting L. pneumophila pneumonia.Conclusions: L. pneumophila incidence is not low in our geographical region in atypical pneumonia patients. Clinicians should consider atypical bacterial pathogens while prescribing antimicrobial management plan.

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Section: Discussionmentioning

confidence: 99%

Molecular Detection of Bacterial Agents of Atypical Pneumonia: Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella pneumophila in Suez Canal region

El-Sayed

2021

Medicine Updates

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“…Finally, 400 μL of supernatant was added to tubes containing 5 μL of carrier RNA, vortexed, then 405 μL of sample with carrier RNA was used for the assay. [7]. Chlamydia pneumoniae, Legionella pneumophila, and Mycoplasma pneumoniae from each bacterial stock were spiked into BAL, using 160 μL of 1x10 5 CFU/ mL stock as input.…”

Section: Specimensmentioning

confidence: 99%

Development of a Real-time PCR assay for Pneumocystis jirovecii on the Luminex ARIES® Platform

Marimuthu¹,

Ghosh²,

Wolf³

2019

JRI

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Pneumocystis pneumonia (PCP) is an opportunistic infection caused by the fungus Pneumocystis jirovecii. Infection with P. jirovecii can result in serious illness in patients with a weakened immune system, and can lead to death if it is not properly diagnosed and treated. Direct detection of P. jirovecii in lower respiratory tract specimens such as bronchoalveolar lavage (BAL) is preferred for rapid diagnosis, a laboratory service currently not available locally. We report here the development of a diagnostic real-time Polymerase Chain Reaction (PCR) assay using BAL specimens to detect P. jirovecii. By targeting the multi-copy mitochondrial large subunit ribosomal RNA gene (mtLSU rRNA) of P. jirovecii, assay sensitivity is increased. Primer pairs were designed to include a fluorescent reporter dye-labeled primer with a unique MultiCode® base pair isoC on the 5'end and one unlabeled primer. The performance characteristics were determined on the Luminex ARIES® instrument, combining DNA extraction, amplification and detection into a one-step process. The cassette contains the reagents needed to perform all of the steps including extraction, purification, amplification, and detection, plus a sample processing control. Accuracy, precision, sensitivity, specificity and stability studies were conducted to validate the assay to meet CLIA requirements. The analytical sensitivity was 89.1%, and the analytical specificity was 100%. The assay could reliably detect 200 organisms/ mL, crossing thresholds (Ct) and melt temperatures (Tm) were consistent, and no cross-reactivity was observed with other pathogens known to cause respiratory infections. The results demonstrated that these primers are specific to Pneumocystis jirovecii. The real-time PCR method using the ARIES® system allowed for rapid and sensitive detection of Pneumocystis pneumonia infections with P. jirovecii using clinical respiratory specimens.

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“…Improved, faster diagnostics that can inform antibiotic choice in hours rather than days can limit the use of inappropriate antibiotics and the associated problems. A variety of approaches have been proposed, but each has limitations; these include nucleic acid- [ 1 , 7 , 8 , 9 , 10 , 11 ], structure- [ 9 , 10 , 12 , 13 ] (e.g., antibody based), mass spectrometry- [ 11 , 14 , 15 , 16 ], fluorescence- [ 17 , 18 , 19 , 20 ], imaging- [ 11 , 21 ], and other optical-based methods [ 22 , 23 , 24 , 25 ]. No ideal method has yet been developed.…”

Section: Introductionmentioning

confidence: 99%

Rapid Detection of Escherichia coli Antibiotic Susceptibility Using Live/Dead Spectrometry for Lytic Agents

et al. 2021

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Antibiotic resistance is a serious threat to public health. The empiric use of the wrong antibiotic occurs due to urgency in treatment combined with slow, culture-based diagnostic techniques. Inappropriate antibiotic choice can promote the development of antibiotic resistance. We investigated live/dead spectrometry using a fluorimeter (Optrode) as a rapid alternative to culture-based techniques through application of the LIVE/DEAD® BacLightTM Bacterial Viability Kit. Killing was detected by the Optrode in near real-time when Escherichia coli was treated with lytic antibiotics—ampicillin and polymyxin B—and stained with SYTO 9 and/or propidium iodide. Antibiotic concentration, bacterial growth phase, and treatment time used affected the efficacy of this detection method. Quantification methods of the lethal action and inhibitory action of the non-lytic antibiotics, ciprofloxacin and chloramphenicol, respectively, remain to be elucidated.

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Real-Time PCR Detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila in Respiratory Specimens Using the ARIES® System

Cited by 3 publications

References 5 publications

Molecular Detection of Bacterial Agents of Atypical Pneumonia: Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella pneumophila in Suez Canal region

Molecular Detection of Bacterial Agents of Atypical Pneumonia: Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella pneumophila in Suez Canal region

Development of a Real-time PCR assay for Pneumocystis jirovecii on the Luminex ARIES® Platform

Rapid Detection of Escherichia coli Antibiotic Susceptibility Using Live/Dead Spectrometry for Lytic Agents

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