Nested polymerase chain reaction (PCR) targeting 16S rDNA for bacterial identification in empyema

Prasad, Rajniti; Kumari, C Sworna; Das, Biswadeep; Nath, Gopal

doi:10.1179/2046905513y.0000000085

Cited by 6 publications

(7 citation statements)

References 5 publications

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“…We found that the multiplex PCR had a better sensitivity and specificity than conventional methods if the analysis is restricted to microorganisms included on the panel. Indeed, when using the clinical final diagnosis of empyema as a reference, the multiplex PCR-based assay had sensitivity six-times higher than that of the (34) No pathological finding 24 (33) Non-purulent secretion 23 (32) Erythema of the mucosa 20 (27) Purulent secretion 17 (23) Endobronchial tumour 8 (11) Bloody secretion 5 (7) conventional microbiologic methods. However, only a minority of the samples and a small percentage of the pathogens diagnosed by conventional methods were included in this panel including bacteria commonly involved in pneumonia.…”

Section: Discussionmentioning

confidence: 99%

“…In previous studies, multiplex PCR methods in different matrices (blood, sputum, throat or nasopharyngeal swabs) have proven to be useful in diagnosing respiratory tract infections and decreasing the financial costs [8]. They have also shown a better sensitivity [9,10], especially in children [11,12]. They also seem to be effective for reducing the incidence of empyema [12].…”

Section: Introductionmentioning

confidence: 99%

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Multiplex bacterial polymerase chain reaction in a cohort of patients with pleural effusion

et al. 2020

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Background: The identification of the pathogens in pleural effusion has mainly relied on conventional bacterial culture or single species polymerase chain reaction (PCR), both with relatively low sensitivity. We investigated the efficacy of a commercially available multiplex bacterial PCR assay developed for pneumonia to identify the pathogens involved in pleural infection, particularly empyema. Methods: A prospective, monocentric, observational study including 194 patients with pleural effusion. Patients were evaluated based on imaging, laboratory values, pleura ultrasound and results of thoracentesis including conventional microbiology studies during hospitalisation. Multiplex bacterial PCR (Curetis Unyvero p55) was performed in batch and had no influence on therapeutic decisions. Results: Overall, there were 51/197 cases with transudate and 146/197 with exudate. In 42% (n = 90/214) there was a clinical suspicion of parapneumonic effusion and the final clinical diagnosis of empyema was made in 29% (n = 61/214) of all cases. The most common microorganisms identified in the cases diagnosed with empyema were anaerobes [31] followed by gram-positive cocci [10] and gram-negative rods [4]. The multiplex PCR assay identified more of the pathogens on the panel than the conventional methods (23.3% (7/30) vs. 6.7% (2/30), p = 0.008). Conclusion: The multiplex PCR-based assay had a higher sensitivity and specificity than conventional microbiology when only the pathogens on the pneumonia panel were taken into account. A dedicated pleural empyema multiplex PCR panel including anaerobes would be needed to cover most common pathogens involved in pleural infection.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Multiplex bacterial polymerase chain reaction in a cohort of patients with pleural effusion

et al. 2020

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“…Empyema pleural fluids contain excess microbial pathogens and bacterial DNA [12,13]. TLR9 acts as a sensor for bacterial DNA which has abundant un-methylated CpG-nucleotides.…”

Section: Discussionmentioning

confidence: 99%

“…The use of CpG-DNA as an anti-inflammatory and anti-infectious immunomodulator and as an adjuvant in immunotherapy has been widely-explored [9,10,11]. Empyema pleural fluids contain high bacterial load and bacterial DNA [12,13] thus during empyema PMCs are constantly exposed to bacterial pathogens and their DNA. However, what impact these will have on PMCs in MRSA empyema is still unknown.…”

Section: Introductionmentioning

confidence: 99%

Heme Oxygenase-1 Deficiency Diminishes Methicillin-Resistant Staphylococcus aureus Clearance Due to Reduced TLR9 Expression in Pleural Mesothelial Cells

et al. 2017

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Methicillin Resistant Staphylococcus aureus (MRSA) cause pneumonia and empyema thoraces. TLR9 activation provides protection against bacterial infections and Heme oxygenase-1 (HO-1) is known to enhance host innate immunity against bacterial infections. However, it is still unclear whether HO-1 regulates TLR-9 expression in the pleura and modulates the host innate defenses during MRSA empyema. In order to determine if HO-1 regulates host innate immune functions via modulating TLR expression, in MRSA empyema, HO-1+/+ and HO-1-/- mouse pleural mesothelial cells (PMCs) were infected with MRSA (1:10, MOI) in the presence or absence of Cobalt Protoporphyrin (CoPP) and Zinc Protoporphyrin (ZnPP) or CORM-2 (a Carbon monoxide donor) and the expression of mTLR9 and mBD14 was assessed by RT-PCR. In vivo, HO-1+/+ and HO-1-/- mice were inoculated with MRSA (5x106 CFU) intra-pleurally and host bacterial load was measured by CFU, and TLR9 expression in the pleura was determined by histochemical-immunostaining. We noticed MRSA inducing differential expression of TLR9 in HO-1+/+ and HO-1 -/- PMCs. In MRSA infected HO-1+/+ PMCs, TLR1, TLR4, and TLR9 expression was several fold higher than MRSA infected HO-1-/- PMCs. Particularly TLR9 expression was very low in MRSA infected HO-1-/- PMCs both in vivo and in vitro. Bacterial clearance was significantly higher in HO-1+/+ PMCs than compared to HO-1-/- PMCs in vitro, and blocking TLR9 activation diminished MRSA clearance significantly. In addition, HO-1-/- mice were unable to clear the MRSA bacterial load in vivo. MRSA induced TLR9 and mBD14 expression was significantly high in HO-1+/+ PMCs and it was dependent on HO-1 activity. Our findings suggest that HO-1 by modulating TLR9 expression in PMCs promotes pleural innate immunity in MRSA empyema.

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“…Ein Empyem ist wahrscheinlich, wenn der pH < 7,0, die Glukose < 40 mg/dl und die LDH > 1,000 IU/l gemessen wird. Die Durchführung einer 16s-rDNA-Untersuchung auf bakterielles Genom mit anschließender Sequenzierung erhöht bei vorbehandelten Patienten die Nachweisrate [128,129]. Eine Zelldifferenzierung des Pleurapunktates (weißes Differenzialblutbild im Ausstrich) kann zu mykobakteriellen Erregern (typischerweise Lymphozytose) und Malignomen (Zellmorphologie) abgrenzen.…”

Section: Lokale Komplikationenunclassified

S2k-Leitlinie Management der ambulant erworbenen Pneumonie bei Kindern und Jugendlichen (pädiatrische ambulant erworbene Pneumonie, pCAP)

et al. 2020

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ZusammenfassungDie vorliegende Fassung der deutschsprachigen AWMF-Leitlinie soll unter Berücksichtigung der vorliegenden Evidenz die medizinische Versorgung von Kindern und Jugendlichen mit ambulant erworbener Pneumonie (pediatric community acquired pneumonia, pCAP) verbessern. In Mitteleuropa steht einer Prävalenz von ca. 300 Fällen pro 100 000 Kinder/Jahr eine sehr geringe Mortalität gegenüber, die Prävention umfasst Hygiene-Maßnahmen und Impfung z. B. gegen Pneumokokken, Hämophilus, Masern und Influenza. Hauptsymptome der pCAP sind Fieber und Tachypnoe, die Diagnosestellung erfolgt primär klinisch durch Anamnese, körperliche Untersuchung und Pulsoxymetrie. Das zusätzliche Vorliegen von Warnsymptomen wie stark reduzierter Allgemeinzustand, Nahrungsverweigerung, Dehydratation, Bewusstseinsstörung oder Krampfanfälle definiert die schwere pCAP in Abgrenzung zur nicht-schweren pCAP. Das Erregerspektrum ist altersabhängig, zur Differenzierung zwischen viraler, bakterieller oder gemischt viral-bakterieller Infektion stehen jedoch keine zuverlässigen Biomarker zur Verfügung. Die meisten Kinder und Jugendlichen mit nicht-schwerer pCAP und O2-Sättigung > 92 % können ohne weitere Röntgen-, Labor- und Erreger-Diagnostik ambulant betreut werden. Der Einsatz von Antiinfektiva ist nicht grundsätzlich indiziert, vor allem bei jungen Kindern, bronchialer Obstruktion und anderen Hinweisen auf virale Genese kann darauf i. d. R. verzichtet werden. Zur kalkulierten Antibiotika-Therapie sind Aminopenicilline Mittel der Wahl, bei gewährleisteter Einnahme und Resorption sind die orale (Amoxicillin) und intravenöse Verabreichung (Ampicillin) von vergleichbarer Wirksamkeit. Nach 48 – 72 Stunden ist eine Verlaufsbeurteilung notwendig, um den Behandlungserfolg und mögliche Komplikationen wie z. B. parapneumonische Ergüsse oder Pleuraempyeme, die eine Erweiterung bzw. Änderung der Therapie erforderlich machen, rechtzeitig zu erfassen.

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Nested polymerase chain reaction (PCR) targeting 16S rDNA for bacterial identification in empyema

Abstract: 16S rDNA PCR improves rates of detection of bacteria in pleural fluid, and can detect bacterial species in a single assay as well as identifying unusual and unexpected causal agents.

Cited by 6 publications

References 5 publications

Multiplex bacterial polymerase chain reaction in a cohort of patients with pleural effusion

Multiplex bacterial polymerase chain reaction in a cohort of patients with pleural effusion

Heme Oxygenase-1 Deficiency Diminishes Methicillin-Resistant Staphylococcus aureus Clearance Due to Reduced TLR9 Expression in Pleural Mesothelial Cells

S2k-Leitlinie Management der ambulant erworbenen Pneumonie bei Kindern und Jugendlichen (pädiatrische ambulant erworbene Pneumonie, pCAP)

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