MD4218C1 - Method for diagnosis of infections caused by enterobacteria producers of beta-lactamases - Google Patents
Method for diagnosis of infections caused by enterobacteria producers of beta-lactamases Download PDFInfo
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- MD4218C1 MD4218C1 MDA20120080A MD20120080A MD4218C1 MD 4218 C1 MD4218 C1 MD 4218C1 MD A20120080 A MDA20120080 A MD A20120080A MD 20120080 A MD20120080 A MD 20120080A MD 4218 C1 MD4218 C1 MD 4218C1
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- 238000000034 method Methods 0.000 title claims abstract description 18
- 102000006635 beta-lactamase Human genes 0.000 title claims abstract description 8
- 241000305071 Enterobacterales Species 0.000 title claims abstract description 5
- 208000015181 infectious disease Diseases 0.000 title claims abstract description 5
- 108020004256 Beta-lactamase Proteins 0.000 title abstract description 4
- 238000003745 diagnosis Methods 0.000 title abstract description 4
- 241000588724 Escherichia coli Species 0.000 claims abstract description 39
- 238000012163 sequencing technique Methods 0.000 claims abstract description 8
- 238000012216 screening Methods 0.000 claims abstract description 7
- 108010002829 beta-lactamase TEM-3 Proteins 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims abstract description 4
- 239000012678 infectious agent Substances 0.000 claims abstract description 3
- 108090000204 Dipeptidase 1 Proteins 0.000 claims description 4
- 238000002405 diagnostic procedure Methods 0.000 claims description 3
- 238000006116 polymerization reaction Methods 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 7
- 230000003321 amplification Effects 0.000 description 5
- 239000003242 anti bacterial agent Substances 0.000 description 5
- 229940088710 antibiotic agent Drugs 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- 230000000844 anti-bacterial effect Effects 0.000 description 4
- 238000000018 DNA microarray Methods 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 238000012790 confirmation Methods 0.000 description 3
- LELAOEBVZLPXAZ-UHFFFAOYSA-N iberin Chemical compound CS(=O)CCCN=C=S LELAOEBVZLPXAZ-UHFFFAOYSA-N 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 208000035473 Communicable disease Diseases 0.000 description 2
- 241000588921 Enterobacteriaceae Species 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 231100000676 disease causative agent Toxicity 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000007403 mPCR Methods 0.000 description 2
- 230000002906 microbiologic effect Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 208000019206 urinary tract infection Diseases 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 239000002132 β-lactam antibiotic Substances 0.000 description 2
- 229940124586 β-lactam antibiotics Drugs 0.000 description 2
- 108091093088 Amplicon Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 150000003952 β-lactams Chemical class 0.000 description 1
Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Invenţia se referă la medicină, în special la un procedeu de diagnosticare a infecţiilor cauzate de enterobacterii producătoare de beta-lactamaze. The invention relates to medicine, in particular to a method for diagnosing infections caused by beta-lactamase-producing enterobacteria.
Actualmente sunt cunoscute diferite teste de diagnosticare microbiologică a agenţilor cauzali ai maladiilor infecţioase şi de caracterizare a acestora. Currently, various tests are known for microbiological diagnosis of the causative agents of infectious diseases and for their characterization.
Este cunoscut testul PCR conventional (Polymerase Chain Reaction sau Reacţie de Polimerizare în Lanţ), care constă în amplificarea enzimatică in vitro a unei secvenţe de ADN de interes. Numărul de copii ale secvenţei ţintă creşte exponenţial cu fiecare ciclu de amplificare, deoarece fiecare secvenţă nucleotidică nou sintetizată constituie o matriţă pentru o nouă copie. Produsul PCR care reprezintă o copie ADN/ARN ţintă este numit amplicon. Acest test permite detectarea cu specificitate înaltă a unor concentraţii scăzute ale secvenţei ţintă [1]. The conventional PCR test (Polymerase Chain Reaction) is known, which consists in the in vitro enzymatic amplification of a DNA sequence of interest. The number of copies of the target sequence increases exponentially with each cycle of amplification, because each newly synthesized nucleotide sequence constitutes a template for a new copy. The PCR product that represents a target DNA/RNA copy is called an amplicon. This test allows the detection of low concentrations of the target sequence with high specificity [1].
Ca tulpină martor pozitiv pentru compararea datelor obţinute în detecţia şi identificarea profilurilor de rezistenţă a bacteriilor la antibioticele betalactamice au fost utilizate tulpinile de E. coli tip CTX-M (CTX-M-1, CTX-M-3, CTX-M-14, CTX-M-15) depozitate în Colecţia Naţională de Microorganisme Nepatogene a Institutului de Microbiologie şi Biotehnologie al AŞM. E. coli strains type CTX-M (CTX-M-1, CTX-M-3, CTX-M-14 , CTX-M-15) stored in the National Collection of Nonpathogenic Microorganisms of the Institute of Microbiology and Biotechnology of the ASM.
Se cunoaşte testul Real Time PCR, care este o variantă a reacţiei PCR şi constă în amplificarea fragmentului ADN în timp real cu analiza acestuia [2]. The Real Time PCR test is known, which is a variant of the PCR reaction and consists in the amplification of the DNA fragment in real time with its analysis [2].
Mai este cunoscută şi Tehnica ADN microarray, care poate fi utilizată la detectarea polimorfismului punctiform de nucleotide (SNPs) sau la detectarea de ADN (studii de hibridizare genomică comparată) sau de ARN (detectat ca ADNc după realizarea revers transcrierii) care poate fi implicat în translarea de proteine [3]. Also known is the DNA microarray technique, which can be used to detect nucleotide point polymorphisms (SNPs) or to detect DNA (comparative genomic hybridization studies) or RNA (detected as cDNA after reverse transcription) that may be involved in protein translation [3].
Este cunoscut, de asemenea, testul de secvenţiere ADN, care reprezintă identificarea tipului, felului şi succesiunii nucleotidelor dintr-un fragment ADN cercetat, unde a fost efectuată amplificarea genelor tulpinii suspecte de producerea de beta-lactamaze cu spectru extins (BLSE) [4]. Rezultatele amplificării supuse secvenţierii sunt comparate cu secvenţele nucleotidice cunoscute. Astfel utilizând secvenţierea este posibilă identificarea nu numai a genelor cunoscute, dar şi a noilor variante cu depozitarea acestora în Colecţia Naţională de Microorganisme Nepatogene care ulterior pot servi ca tulpini de referinţă în determinarea markerilor de rezistenţă la antibioticele betalactamice (BLSE). The DNA sequencing test is also known, which represents the identification of the type, kind and sequence of nucleotides from a researched DNA fragment, where the amplification of the genes of the strain suspected of producing extended-spectrum beta-lactamase (ESBL) was performed [4] . The results of the amplification subjected to sequencing are compared with the known nucleotide sequences. Thus, using sequencing, it is possible to identify not only known genes, but also new variants with their storage in the National Collection of Nonpathogenic Microorganisms, which can later serve as reference strains in the determination of resistance markers to beta-lactam antibiotics (BLSE).
Cea mai apropiată soluţie de procedeul propus este procedeul de determinare a rezistenţei la antibiotice (screening a BLSE) a bacililor gram-negativi cu utilizarea testelor de diagnosticare microbiologică a agenţilor cauzali ai maladiilor infecţioase şi de caracterizare a acestora, şi anume: PCR convenţional sau PCR Real Time (8 ore) pentru screening şi tehnica ADN microarray (24 ore) sau secvenţierea (12 ore) pentru confirmare, unde procedeul durează 20…32 ore [5]. The closest solution to the proposed procedure is the procedure for determining the resistance to antibiotics (screening of BLSE) of gram-negative bacilli with the use of microbiological diagnostic tests of the causative agents of infectious diseases and their characterization, namely: conventional PCR or PCR Real Time (8 hours) for screening and the DNA microarray technique (24 hours) or sequencing (12 hours) for confirmation, where the procedure takes 20...32 hours [5].
Dezavantajul acestor teste constă în timpul îndelungat de determinare şi caracterizare a microorganismelor rezistente la antibiotice şi de determinare a profilului BLSE ce împiedică prescrierea precoce a preparatului antibacterian etiotrop. The disadvantage of these tests is the long time required to determine and characterize antibiotic-resistant microorganisms and to determine the BLSE profile, which prevents the early prescription of the etiotropic antibacterial preparation.
Problema pe care o soluţionează invenţia propusă constă în elaborarea unui procedeu de depistare rapidă a microorganismelor din familia Enterobacteriaceae producente de beta-lactamaze şi profilului BLSE. The problem that the proposed invention solves consists in the development of a procedure for rapid detection of microorganisms from the Enterobacteriaceae family producing beta-lactamases and the BLSE profile.
Esenţa invenţiei constă în faptul că se propune un procedeu de diagnosticare a infecţiilor cauzate de enterobacterii producătoare de beta-lactamaze care constă în aceea că se efectuează screeningul prin metoda multiplex al reacţiei de polimerizare în lanţ cu utilizarea tulpinilor de referinţă (E. coli profil BLSE CTX-M-1 din grupul filogenetic A, E. coli profil BLSE CTX-M-3 din grupul filogenetic A, E. coli profil BLSE CTX-M-3 din grupul filogenetic B2, E. coli profil BLSE CTX-M-14 din grupul filogenetic A, E. coli profil BLSE CTX-M-14 din grupul filogenetic B2, E. coli profil BLSE CTX-M-14 din grupul filogenetic D, E. coli profil BLSE CTX-M-15 din grupul filogenetic A, E. coli profil BLSE CTX-M-15 din grupul filogenetic B2, E. coli profil BLSE CTX-M-15 din grupul filogenetic D), iar în cazul stabilirii unor noi tulpini se efectuează metoda de secvenţiere pentru stabilirea agentului infecţios şi includerea lui în lista tulpinilor de referinţă. The essence of the invention consists in the fact that a diagnostic procedure for infections caused by beta-lactamase-producing enterobacteria is proposed, which consists in performing screening by the multiplex method of the polymerization chain reaction using reference strains (E. coli BLSE profile CTX-M-1 from phylogenetic group A, E. coli BLSE profile CTX-M-3 from phylogenetic group A, E. coli BLSE profile CTX-M-3 from phylogenetic group B2, E. coli BLSE profile CTX-M-14 from phylogenetic group A, E. coli BLSE profile CTX-M-14 from phylogenetic group B2, E. coli BLSE profile CTX-M-14 from phylogenetic group D, E. coli BLSE profile CTX-M-15 from phylogenetic group A, E. coli BLSE profile CTX-M-15 from phylogenetic group B2, E. coli BLSE profile CTX-M-15 from phylogenetic group D), and in the case of establishing new strains, the sequencing method is performed to establish the infectious agent and include it in the list of reference strains.
Rezultatul invenţiei constă în reducerea timpului de depistare a bacteriilor rezistente la preparatele antibacteriene şi definitivarea rezultatului peste 3…4 ore, iar în cazul tulpinilor nontipabile rezultatul poate fi definitivat peste 15…16 ore. The result of the invention consists in reducing the detection time of bacteria resistant to antibacterial preparations and finalizing the result in 3...4 hours, and in the case of non-typable strains, the result can be finalized in 15...16 hours.
Exemplu de realizare a invenţiei Example of realization of the invention
Procedeul dat a fost utilizat la testarea sensibilităţii la antibiotice a 196 tulpini de E. coli izolate din probe clinice (urină şi mase fecale), colectate de la persoane cu diagnostic de infecţii ale căilor urinare internate în IMSP Institutul de Cercetări Ştiinţifice din Domeniul Ocrotirii Sănătăţii Mamei şi Copilului, IMSP SCM „Sfânta Treime” şi IMSP Spitalul Clinic Municipal nr.1. La persoanele implicate în studiu s-a efectuat o analiză citobacteriologică a urinei şi s-a colectat anamneza epidemiologică. La majoritatea persoanelor au fost prezente antecedente ale infecţiilor urinare - în jur de 97%. Vârsta pacienţilor a fost variată, de la 18 până la 42 ani. Din lotul pacienţilor investigaţi femeile au fost mai des afectate decât bărbaţii, ponderea acestora constituind 85% din lotul cercetat. Datele anamnezei epidemiologice au demonstrat că o mare parte din pacienţi au utilizat antibiotice înainte de a fi incluşi în studiu. Rezistenţa tulpinilor E. coli la preparatele antimicrobiene betalactamice a fost determinată şi prin metode de biologie moleculară cu utilizarea tulpinilor tip cu profil BLSE cunoscut. Spre deosebire de metodele de biologie moleculară clasice - PCR conventional sau PCR Real Time, aplicarea metodei PCR Multiplex cu utilizarea tulpinilor tip cu profil BLSE cunoscut permite cu specificitate şi sensibilitate înaltă evidenţierea relaţiei dintre informaţia genetică şi expresia acesteia pentru prescrierea precoce a tratamentului antibacterian şi prevenirea consumul neargumentat de antibiotice. The given procedure was used to test the sensitivity to antibiotics of 196 strains of E. coli isolated from clinical samples (urine and faeces), collected from people with a diagnosis of urinary tract infections hospitalized in the IMSP Institute for Scientific Research in the Health Care Field Mother and Child, IMSP SCM "Holy Trinity" and IMSP Municipal Clinical Hospital no. 1. For the people involved in the study, a cytobacteriological analysis of the urine was performed and the epidemiological anamnesis was collected. Most of the people had a history of urinary infections - around 97%. The age of the patients was varied, from 18 to 42 years. From the group of investigated patients, women were more often affected than men, their weight constituting 85% of the researched group. Epidemiological anamnesis data demonstrated that a large proportion of patients used antibiotics before being included in the study. The resistance of E. coli strains to beta-lactam antimicrobial preparations was also determined by molecular biology methods using type strains with a known BLSE profile. Unlike the classical molecular biology methods - conventional PCR or Real Time PCR, the application of the Multiplex PCR method with the use of type strains with a known BLSE profile allows with high specificity and sensitivity to highlight the relationship between genetic information and its expression for the early prescription of antibacterial treatment and prevention unreasonable use of antibiotics.
Eficienţa invenţiei propuse faţă de cea mai apropiată soluţie pentru detectarea BLSE The efficiency of the proposed invention compared to the closest solution for BLSE detection
Cea mai apropiată soluţie Durata (ore) Invenţia Durata (ore) Screening: PCR conventional sau PCR Real Time 8 Screening: PCR Multiplex cu utilizarea tulpinilor de referinţă (E. coli tip profil BLSE CTX-M-1, grup filogenetic A; E. coli tip profil BLSE CTX-M-3, grup filogenetic A; E. coli tip profil BLSE CTX-M-3, grup filogenetic B2; E. coli tip profil BLSE CTX-M-14, grup filogenetic A; E. coli tip profil BLSE CTX-M-14, grup filogenetic B2; E. coli tip profil BLSE CTX-M-14, grup filogenetic D; E. coli tip profil BLSE CTX-M-15, grup filogenetic A; E. coli tip profil BLSE CTX-M-15, grup filogenetic B2; E. coli tip profil BLSE CTX-M-15, grup filogenetic D) 3…4 confirmarea: Tehnica ADN microarray sau secvenţierea 24 12 confirmarea: secvenţierea în cazul depistării tulpinilor non-tipabile 12 Total 20…32 3…16 The closest solution Duration (hours) Invention Duration (hours) Screening: conventional PCR or Real Time PCR 8 Screening: Multiplex PCR using reference strains (E. coli profile type BLSE CTX-M-1, phylogenetic group A; E. coli BLSE profile type CTX-M-3, phylogenetic group A; E. coli BLSE profile type CTX-M-3, phylogenetic group B2; E. coli BLSE profile type CTX-M-14, phylogenetic group A; E. coli type BLSE profile CTX-M-14, phylogenetic group B2; E. coli BLSE profile type CTX-M-14, phylogenetic group D; E. coli BLSE profile type CTX-M-15, phylogenetic group A; E. coli BLSE profile type CTX-M-15, phylogenetic group B2; E. coli BLSE profile type CTX-M-15, phylogenetic group D) 3...4 confirmation: DNA microarray technique or sequencing 24 12 confirmation: sequencing in case of detection of non-typeable strains 12 Total 20...32 3...16
Astfel, utilizarea procedeului algoritmizat nou de depistare a producerii beta-lactamazelor la microorganismele familiei Enterobacteriaceae, prin aplicarea tulpinilor tip profil BLSE, permite identificarea şi caracterizarea rapidă a genotipului care codifică exprimarea profilurilor de rezistenţă la antibioticele betalactamice şi duce la eficientizarea sistemului de monitorizare şi supraveghere a rezistenţei la antibiotice, iar, ulterior, aceasta va perfecţiona tactica de prescriere a tratamentului antibacterian şi va preveni consumul neargumentat de antibiotice. Thus, the use of the new algorithmized procedure for detecting the production of beta-lactamases in microorganisms of the Enterobacteriaceae family, by applying the BLSE profile type strains, allows the rapid identification and characterization of the genotype that encodes the expression of resistance profiles to beta-lactam antibiotics and leads to the efficiency of the monitoring and surveillance system of antibiotic resistance, and subsequently, this will improve the tactic of prescribing antibacterial treatment and prevent the unjustified consumption of antibiotics.
1. Bernards A.T., et. al. NVMM guideline laboratory detection of highly resistant microorganisms, ver. 1.0, 2011, p.23 1. Bernards A.T., et. al. NVMM guideline laboratory detection of highly resistant microorganisms, ver. 1.0, 2011, p.23
2. Bernards A.T., et. al. NVMM guideline laboratory detection of highly resistant microorganisms, ver. 1.0, 2011, p.25 2. Bernards A.T., et. al. NVMM guideline laboratory detection of highly resistant microorganisms, ver. 1.0, 2011, p.25
3. Cohen Stuart J., et. al. Rapid detection of TEM, SHV and CTX-M extended spectrum beta-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis. Journal of Antimicrobial Chemotherapy, 2010, vol. 65, p 1377-1381 3. Cohen Stuart J., et. al. Rapid detection of TEM, SHV and CTX-M extended spectrum beta-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis. Journal of Antimicrobial Chemotherapy, 2010, vol. 65, p 1377-1381
4. Bernards A.T., et. al. NVMM guideline laboratory detection of highly resistant microorganisms, ver. 1.0, 2011, p.19 4. Bernards A.T., et. al. NVMM guideline laboratory detection of highly resistant microorganisms, ver. 1.0, 2011, p.19
5. Bernards A.T., et. al. NVMM guideline laboratory detection of highly resistant microorganisms, ver. 1.0, 2011, p.28 5. Bernards A.T., et. al. NVMM guideline laboratory detection of highly resistant microorganisms, ver. 1.0, 2011, p.28
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