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Biologia molecular: Una necessitat de la microbiologia clàssica
Jordi Vila Hospital Clinic
DIAGNOSTIC TECHNOLOGIES IN CLINICAL MICROBIOLOGY
• Direct examination - • Culture
Microscopy
• Detection of antibodies (serology) • Detection of microbial antigens • Detection of nucleic acids
Main features for an optimal test
• • • • •
Detection and identification of several microorganisms related to a specific syndrome in a single test High sensitivity and specificity Rapid, in minutes better than hours Automatized Cheap
Rapid diagnostic methods
• Adventages – To implement adequate antimicrobial therapy – To favor the control of the emergence of
antimicrobial resistance
– Decrease the cost to the hospital
EVOLUTION OF THESE MOLECULAR TOOLS
• Hybridization of nucleic acids • DNA or RNA amplification • Real time PCR • DNA microarrays
TYPES OF NUCLEIC ACID AMPLIFICATION
• Target amplification system – PCR – Ligase chain reaction (LCR) – Self-sustaining sequence amplification (3SR) – Nucleic acid sequence-based amplification (NASBA) – Transcription-mediated amplification (TMA) – Strand displacement amplification (SDA) • Signal amplification system – Q -b-
replicase
– Branched DNA technologies (bDNA) – Cleavage-based signal amplification
VARIATIONS OF THE PCR
• Multiplex PCR • Nested PCR • RT-PCR • Broad-range PCR • Real-time PCR • Arbitrarily primed PCR
Broad-range PCR
P1 P2 16S ITS 23S 5S Consensus Regions Variable Regions ITS = Internal transcribed spacer 798 bp
APPLICATIONS 0F 16S rRNA
• From colony: – Bacteria with difficult phenotypic identification • Species of Acinetobacter, Corynebacterium, some anaerobes – Fastidious bacteria, due to nutritional requirements • Eikenella corrodens, Nocardia spp., etc. – Slow growth bacteria • Non-tuberculosis Mycobacteria – Bacteria with no phenotypic match with known bacteria • From clinical samples: – Previously treated with antibiotics (CSF, amniotic fluid, joint fluid, cardiac
valves)
• When culture is negative – Non-cultured bacteria • Rochalimae (Bartonella) quintana
VARIATIONS OF THE PCR
• Multiplex PCR • Nested PCR • RT-PCR • Broad-range PCR • Real-time PCR • Arbitrarily primed PCR
Real time PCR
(specific probes) Primer 3’ 5’ TaqMan probes A = Reporter dye B = Quenching dye A B Primer 3’ 5’ Molecular beacons A B Primer 3’ 5’ FRET Probes
Real time PCR
• Advantages: – Speed (1-3 hours) – Closed system decreasing the risk of contamination – Quantitation of the initial
nucleic acid
APPLICATIONS
Detection of toxins
• Detection of C. difficile toxin • Detection of PVL in S. aureus • Detection of virulence factors in diarrhoeagenic
E.coli (Multiplex PCR directly from a colony identified as E.coli)
– Enterotoxigenic E.coli – Enteroaggregative E.coli – Enteropathogenic E.coli
APPLICATIONS
Detection of specific microorganisms
• Grow slowly, or for which the cultivation methods
are not widely available or do not exist .
– Examples: – Bartonella spp. – Borrelia burgdorferi – Ricketssias, Ehrlichia and Coxiella spp. – Mycoplasma pneumoniae – Chlamydophila pneumoniae – Bordetella pertussis – Tropheryma whipplei
APPLICATIONS
Detection of specific virus Detection
– Parainfluenza virus 1,2,3 and 4 – Influenza virus A, B and C – Adenovirus – Respiratory syncytial virus A and B – Coronavirus – Herpes simple virus – Human papillomavirus – Enterovirus
Viral load
– HIV – HBV – HCV – CMV – EBV
APPLICATIONS
• Where reliable and rapid detection of infected or
colonized patients and health care workers is used for minimizing spread of antimicrobial resistant bacteria in health care institution.
Ex. MRSA
• Detection of
S. agalactiae
in vaginal swabs.
Detection and identification of several microorganisms in a single test
• Respiratory infections • Gastrointestinal infections • Sexually transmited diseases infection
Current alternatives to detect respiratory virus
• Detection of specific microorganisms. – Virus – Parainfluenza virus 1,2,3 and 4 – Influenza virus A, B and C – Adenovirus – Respiratory syncytial virus A and B – Coronavirus – Enterovirus – Metapneumovirus
5’ 5’ 5’ 5’ N IVB ADV IVB RSV IVA RSV + ADV Nested-RT-PCR
Current alternatives to detect respiratory virus
• Detection of specific microorganisms. – Filmarray – Magicplex-RV (Seegene) – Abbot – Luminex – Resplex (Qiagen) – Most of them are detecting the most prevalent respiratory virus some of
them can also detect atypical bacteria causing pneumonia
– Overall good sensitivity and specificity
Classical Blood culture Gram stain Culture ID/Antibiogram Blood Semi-molecular Blood culture Gram stain Identification/Resistance - DNA Miroarrays - Filmarray
Microarrays de ADN
120 sondas para identificar:
Pseudomonas aeruginosa Staphylococcus aureus Escherichia coli
Genes: Housekeeping Virulencia Resistencia
gen aac-aphD gen cat gen dfrS1 gen ermA gen mecA
Tiempo total 8h.
JCM (2006) 44:2389
Classical Blood culture Gram stain Culture ID/Antibiogram Blood Semi-molecular Blood culture Gram Stain Culture ID/Anti Identification/Resistance MS DNA Miroarrays Filmarray
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) Acceleration Drift Matrix-embedded Analyte on Microtitreplate Target + + + Electrodes Laser Desorption/Ionization + + + Detector Time-of-Flight Intensity m/z
MALDI-TOF MS
Identified species Bacillus globigii BioProfiler Data interpretation Generate MALDI-TOF profile spectrum Smear a thin-layer onto a MALDI target plate Select a colony Unknown microrganism ?
a.i.
6000 4000 2000 0 -2000 -4000 -6000
Profiling results from different Bacillus strains B. globigii B. licheniformis B. subtilis B. thuringiensis
Direct testing of positive blood cultures by MALDI-TOF
WHEN POSITIVE Sampling Incubation of blood culture bottles Preparation of a bacterial pellet Comparison with a database Acquisition of the proteic profile Deposition of bacterial pellet on MALDI microplate
%
Identification of bacteria growth in blood cultures
70 97 93 97 Juiz et al. EJCM (2011) “in press”
Burckhardt and Zimmerman: Using matrix-assisted laser desoprtion ionization-time of flight mass spectrometry to detect carbapenem resistance within 1 to 2.5 hours Journal of Clinical Microbiology 2011; 49: 3321
• 10 mcl loopful of bacteria to 1 ml of 0.45% NaCl with or without
0.5 g/liter ertapenem. Incubation 2.5 h at 36ºC This methods works for strains carrying NDM-1, VIM-1, VIM-2, KPC-2 and different IMP enzymes
Classical Blood culture Gram stain Culture ID/Antibiograma Blood Semi-molecular Blood culture Gram Stain Identification/Resistance MS Genexpert (MRSA) Miroarrays de ADN Filmarray
Parta M et al.
Identification of methicillin-resistant or methicillin susceptible Staphylococcus aureus in blood cultures and wound swabs by GeneXpert.
J Clin Microbiol 2009;47:1609
• 223 blood cultures – 68 positive to S. aureus – 47 MRSA and 21 MSSA – PCR 46/47 (98%) MRSA and 21/21 MSSA (100%)
Clasical Blood culture Gram Stain Culture ID/Antibiogram Blood Semi-molecular Blood culture Molecular Direct detection - Septifast - Magicplex-sepsis - SeptiTest Gram Stain Culture ID/Anti Identification/Resistance MS Genexpert (MRSA) Miroarrays de ADN Filmarray
CONCLUSIONS
Advantages of these rapid tests compared to BC: - Patients receiving antibiotic - Detection of fungemia caused mainly by Aspergillus - More rapid identification than the BC.
Advantages of the BC compared to these rapid tests: - Possibility to determine antimicrobial susceptibility - Detect microorganisms not included in the rapid test
PCR and Mass Spectrometry
(Detection of nucleic acids)
• SEQUENOM
– Amplification / Transcription / Cleavage / Detection of
restriction fragment by MALDI-TOF
• ESI Mass Spectrometry (PLEX-ID)
ESI-TOF Mass Spectrometry
Sprayer Dry Gas Heater Q- Separation CID Dual Ion Funnel Hexapole TOF-MS Collision Gas Supply Glass Capillary Reflectron Flight Tube Analytical Quadrupole Collision Cell Orthogonal Accelerator Detector API Spray Chamber
PCR/ESI Mass Spectrometry
• First, broad-range primers, targeting sites that are highly conserved
in all members of a microbe family, are used to amplify PCR products from groupings of microbes rather than single species.
• These primers are coupled with species- or strain-specific primers
for the identification of specific pathogens or antibiotic targets.
• Second, PCR conditions are, by design, permissive and thus tolerant
of mismatches, so that even sequences from novel strains can be amplified.
• Third, inosine and other “wild-card” nucleotides are used in primers
to facilitate PCR analysis of mispaired sequences.
PCR/ESI Mass Spectrometry
• Fourth, because MS simply measures the mass-to-charge ratio (m/z),
the sequence of the amplicon need not be known in order to detect it. The technology offers advantages over routine single-target and multiplex PCR in that it is a full bioinformatics sequence analysis system.
• After amplification, MS is used to rapidly determine the precise
mass-to-charge ratio for the amplified nucleic acid fragments present, and the A, C, T, and G contents (i.e., the base composition) of each amplicon are determined using proprietary software that creates a signature to allow organism identification and genotyping.
• This novel MS technology enables the rapid, sensitive, cost-effective,
and simultaneous detection of a wide range of typical pathogenic organisms.
PCR/ESI Mass Spectrometry
• Broad amplification of all microorganisms – Bacteria, virus, fungi and protozoos • Rapid detection-identification (6-8 hours) • High throughput • Able to detect co-infections • Quantitative • Detect new pathogens
Next generation DNA sequencing
• Full genome sequence • 1-2 weeks • All genomic information for a specific
microorganism
Next generation DNA sequencing
• Intrahost variability of HIV or HBV – Tropism – Antiviral resistance • Compartmentalization of viral quasispecies • Viral dynamics – During natural history – After therapeutic intervention
Do you think that molecular tools will fully replace conventional bacteriology?
MB I DO NOT THINK SO