Comparative effectiveness of molecular tests based on isothermal amplification and polymerase chain reaction for detection carbapenemase genes in hospital-acquired pneumonia

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BACKGROUND: Antibiotic resistance in pathogens causing hospital-acquired pneumonia (HAP), particularly to carbapenems non-susceptibility, is a serious public health problem. New rapid and sensitive diagnostic methods are required for the timely initiation of effective antimicrobial therapy. The development and implementation of molecular diagnostic tests, based on polymerase chain reaction and promising isothermal amplification techniques, will significantly reduce turnaround time and improve detection of HAP pathogens with carbapenemase genes.

AIM: The aim was to compare efficacy of a new loop-mediated isothermal amplification (LAMP) test for the detection of NDM, OXA-48, and KPC carbapenemase genes and routine laboratory tests based on real-time polymerase chain reaction in the diagnosis of hospital-acquired lower respiratory tract infections (LRTI), including hospital-acquired pneumonia.

MATERIALS AND METHODS: The study included 141 samples of lower respiratory tract biomaterial collected from 107 patients with hospital-acquired LRTI (hospital-acquired pneumonia and/or purulent tracheobronchitis). Tracheal aspirate (TA, n=78), bronchoalveolar lavage (BAL, n=29) and sputum (n=34) were used as biomaterial samples. Nucleic acids were isolated using an AmpliTest® RIBO-prep kit. NDM, OXA-48 and KPC genes were analyzed using the proposed AmpliTest® CP NDM/OXA-48/KPC LAMP reagent kit by loop isothermal amplification (LAMP). To compare results, real-time PCR was performed using the AmpliSens MDR MBL-FL and AmpliSens MDR KPC/OXA-48-FL reagent kits.

RESULTS: Concordant results were obtained for the detection of NDM, OXA-48 and KPC carbapenemase genes in lower respiratory tract biomaterial samples using a new LAMP test and PCR tests for all samples tested. Based on the time to positivity (TTP), the turnaround time for LAMP was shorter than the turnaround time for PCR. Additional PCR tests were used to identify the Gram-negative microorganisms in the samples tested (both individually and in combination).

CONCLUSION: The obtained data showed that the efficiency of detection of the study groups of carbapenemase genes by the new LAMP test is not lower than the efficiency of their detection by the PCR tests previously used in practice.

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作者简介

Danilov Dmitry Igorevich Danilov

Centre for Strategic Planning and Management of Biomedical Health Risks

编辑信件的主要联系方式.
Email: danilov.i.dmitry@gmail.com
ORCID iD: 0000-0001-5772-8498
SPIN 代码: 8372-8456
俄罗斯联邦, 10 bldg. 1 Pogodinskaya street, 119121 Moscow

Elizaveta Glushchenko

Centre for Strategic Planning and Management of Biomedical Health Risks

Email: Glushhenko@cspfmba.ru
ORCID iD: 0000-0002-6674-2045
SPIN 代码: 7440-4930
俄罗斯联邦, 10 bldg. 1 Pogodinskaya street, 119121 Moscow

Yulia Savochkina

Centre for Strategic Planning and Management of Biomedical Health Risks

Email: YSavochkina@cspmz.ru
ORCID iD: 0000-0003-2313-0521

Cand. Sci. (Biology)

俄罗斯联邦, 10 bldg. 1 Pogodinskaya street, 119121 Moscow

Daria Strelkova

Sechenov First Moscow State Medical University (Sechenov University)

Email: dashastrelkova@gmail.com
ORCID iD: 0000-0002-2124-0623
SPIN 代码: 9549-8053

MD, Cand. Sci. (Medicine)

俄罗斯联邦, Moscow

Svetlana Rachina

Sechenov First Moscow State Medical University (Sechenov University)

Email: rachina_s_a@staff.sechenov.ru
ORCID iD: 0000-0002-3329-7846
SPIN 代码: 1075-7329

MD, Dr. Sci. (Medicine), Assistant Professor

俄罗斯联邦, Moscow

Diana Gasanova

Sechenov First Moscow State Medical University (Sechenov University)

Email: gasanova_556@mail.com
ORCID iD: 0009-0008-9830-7516
俄罗斯联邦, Moscow

Lyudmila Fedina

Moscow City Hospital named after S.S. Yudin

Email: fedina201368@gmail.com
ORCID iD: 0000-0002-6417-9535
SPIN 代码: 1961-7486
俄罗斯联邦, Moscow

Igor Sychev

Moscow City Hospital named after S.S. Yudin

Email: sychevigor@mail.ru
ORCID iD: 0000-0002-2970-3442
SPIN 代码: 7282-6014

MD, Cand. Sci. (Medicine), Assistant Professor

俄罗斯联邦, Moscow

Egor Larin

Hospital for War Veterans No. 3

Email: dr.egorlarin@gmail.com
ORCID iD: 0000-0002-7450-6317
俄罗斯联邦, Moscow

German Shipulin

Centre for Strategic Planning and Management of Biomedical Health Risks

Email: Shipulin@cspfmba.ru
ORCID iD: 0000-0002-3668-6601
SPIN 代码: 1908-9098

MD, Cand. Sci. (Medicine)

俄罗斯联邦, 10 bldg. 1 Pogodinskaya street, 119121 Moscow

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2. Fig. 1. Examples of fluorescence signal graphs for the detection of carbapenemase genes in control samples with target DNA concentrations of 1x10^5 copies/mL, 1x10^4 copies/mL, and 1x10^3 copies/mL, as well as the detection of the internal control sample: a) detection of MBL genes from the NDM group (signal in the HEX channel in the first reaction mixture); b) detection of IC DNA (signal in the ROX channel in the first mixture); c) detection of carbapenemase genes from the KPC group (signal in the HEX channel in the second reaction mixture); d) detection of carbapenemase genes from the OXA-48 group (signal in the ROX channel in the second reaction mixture).

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3. Fig. 2. TTP diagram for PCR-RT and LAMP methods in the analysis of lower respiratory tract clinical samples within the study: a) PCR-RT TTP for the OXA-48, NDM, and KPC target genes; b) LAMP TTP for the OXA-48, NDM, and KPC target genes.

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版权所有 © Danilov D.I., Glushchenko E.E., Savochkina Y.A., Strelkova D.A., Rachina S.A., Gasanova D.R., Fedina L.V., Sychev I.N., Larin E.S., Shipulin G.A., 2025

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