Diagnosis of Infection (PDF)

Diagnosis of Infection Professor Michael Prentice 1 Lecture objectives Describe the importance of correct specimen labelling and packaging by clinicians: avoiding errors,providing clinical information e.g. foreign travel history reducing infection risks to staff) Name the most common microbiology sp...

Diagnosis of Infection Professor Michael Prentice 1 Lecture objectives Describe the importance of correct specimen labelling and packaging by clinicians: avoiding errors,providing clinical information e.g. foreign travel history reducing infection risks to staff) Name the most common microbiology specimens urines, swabs, be aware that that MSU is not the generic name for all urine samples Name less common, more significant samples and understand the difference between sterile and non-sterile sites Sterile sites: blood, csf,. Non routine Bronchoalveolar lavage or BAL etc, Discern the difference between rapid initial tests such as microscopy , Gram staining and culture based tests in terms of time taken and specificity of result Be aware of modern advances in laboratory diagnosis such as Molecular diagnosis , MALDI-TOF Formulate appropriate specimen requests to diagnose specific clinical conditions 2 Diagnosis of Infection Specimens: basis of laboratory diagnosis (Pre-analytical) Labelled specimen in bag with outer pocket for request form Labelled request form with patient identifier and diagnostic details. Request form often computer generated Inform lab if particular infection risk General principle: garbage in garbage out – must label and send appropriate specimen. Laboratories generally offer a handbook to help this 3 Sources of Laboratory Test Errors Pre-analytical Analytical Post-analytical Specimen acquisition, identification, handling, transport, pre-test processing Testing process Interpretation of results in making a diagnosis Most likely error source Least likely error source (GIGO Garbage in – Garbage out) 4 Special considerations for Microbiology Specimens Successful culture amplifies pathogen numbers We have systems to handle this but some pathogens, uncommon locally, are notorious in ability to infect Microbiology laboratory workers Brucella species (brucellosis) travel to Eastern Mediterranean- Turkey, Lebanon Salmonella enterica Serovar Typhi (typhoid fever): travel to Indian subcontinent – India, Pakistan, Nepal Coccidioides immitis, Histoplasma (Dimorphic Fungi) travel to N/S America, Africa Essential to specify foreign travel if sending (blood, urine, csf) cultures from patients 5 Diagnostic microbiology Microscopy: can be a same day activity Unfixed specimens – urine for cells and bacteria Fixed specimens – Gram and Ziehl Neelsen stain (mycobacteria) Fluorescence microscopy Immunofluorescence Auramine (mycobacteria) Electron microscopy – historic virus diagnosis Culture: takes at least one day solid agar media (colonies) Broth (turbidity, more sensitive than agar medium) Essential for sensitivity testing (takes one day after initial culture i.e. generally 2 days) Serology (antibody and antigen based): often batched 1-2 times per week Molecular methods (DNA, RNA dependent) can be same day for proprietary kits Polymerase chain reaction Genome or metagenome sequencing (DNA probes) 6 Direct microscopy Light microscopy Unstained preparations Simple stains - Gram, Giemsa Special stains - Ziehl-Nielson, India ink Immunofluorescence Based on specific antibody Electron microscopy Previously extensively used for virology diagnosis, not now 7 Stained bacteria Staphylococcus aureus Gram positive cocci Mixed Gram positive cocci + Gram negative coccobacilli (Streptococcus pneumoniae + Haemophilus influenzae) Ziehl-Neelsen Stain (Mycobacterium species and Staphylococcus epidermidis) Gram negative bacilli (Escherichia coli ) 8 Bacterial Growth Solid or liquid media Agar plates, slopes, broth culture Atmosphere: Aerobic, anaerobic or microaerophilic Facultative or obligate anaerobes Usually at 37 °C Most clinically important bacteria grow overnight, or within a few days Mycobacteria can take months Some can not be grown 9 Swab technology Wound and mucosal surface swab For Gram stain and bacterial culture Transport medium to maintain bacterial viability Rayon swab soft and absorbent fibre spun from wood pulp 10 Flocked and rayon swab designs. Peter Daley et al. J. Clin. Microbiol. 2006; doi:10.1128/JCM.02055-05 Flocked swab: better at sampling host cells and mucus from mucosal surfaces e.g. NPA Paired with proprietary molecular transport media (MTM) or nucleic acid transport (NAT) or Inactivating Viral Transport Medium contains guanidine thiocyanate (GCN) GCN preserves nucleic acid and inactivates viruses (safer if spilled) 11 swabs Research evidence self-collected saliva may be as good a sample for SARS-CO-V2 as nasal Viral transport medium contains bovine serum, buffered salts and antibiotics maintains virus viability for later cell culture Some types may also transport viable bacteria needing cell culture Chlamydia Mycoplasma Brownish-red Indicates likely BSA Potential infection risk if spilled Most SARS-CoV2 swabs in Molecular Transport Media/ Inactivating Viral Transport Medium 12 Other specimens Sputum Urine Faeces Pus (better than swab for anaerobes) CSF from lumbar puncture EDTA blood (DNA, full blood counts) Clotted blood (Serology, cross match) Blood Culture Sterile samples: any isolate potentially significant (contamination still possible) 13 End of Part 1 True-False Questions 1. Most testing errors occur during laboratory analysis 2. Any bacterium isolated from faeces is significant 3. Gram stain takes several days to perform 14 Part 2: Answers to Part 1 TrueFalse Questions 1. Most testing errors occur during laboratory analysis FALSE 2. Any bacterium isolated from faeces is significant FALSE 3. Gram stain takes several days to perform FALSE 15 Diagnostic modalities for infecting pathogens Gram stain Biochemical reactions, serology specimen Real time PCR (DNA/RNA based) Conventional culture MALDI-TOF 16 (protein based) Blood culture technology (See podcast) A Phlebotomy+ inoculation B http://www.pc.maricopa.edu/healthenhancement/Phlebotomy/phleb_new.jpg C incubation Similar machines now used for Mycobacteria (TB) culture Production of carbon dioxide turns sensor yellow. 17 Pure culture of blood culture bottle contents Requires solid culture media (not liquid broth) Inoculating loop 1881 Frau Lina Hesse 18 Laboratory reports on the 8-10% of blood cultures which are positive for bacteria Day 1, Gram stain “Gram-negative rods seen in blood culture broth” – i.. indicative but not specific. PCR for specific pathogens possible Day 2 Growth characteristics on agar Biochemical profile (API) or MALDI-TOF identity from colonies on agar , serology, some antimicrobial sensitivity “lactose fermenting coliform/Klebsiella pneumoniae isolated sensitive to ampicillin” Skin organisms grown e.g. Staphylococcus epidermidispossible contaminants Day 3 more sensitivities “Klebsiella pneumoniae isolated , sensitive to ciprofloxacin, gentamicin etc” 19 Diagnosis of bacteraemia Blood culture liquid system sensitivity of 1 CFU/ml level bacteraemia greater in children problems with contamination (organisms from skin) molecular techniques also applicable 20 MALDI-TOF (Mass Spectroscopy) and bacterial identification 21 Sensitivity Testing Antibiotic impregnated Disc Zone of inhibition Rate of growth in broth (proprietary Vitek2 system) http://2017.igem.org/Team:Sheffield/Human_Practices Can see mixtures Mixtures of bacteria invalidate test 22 21st Century Microbial technology Broth/agar bacterial culture and Gram stain still common Rapid protein bacterial identification methods (MALDI-TOF) and DNA based methods (PCR) coming in Virology predominantly nucleic acid/antibody based 23 Question: Why do we still use agar/Gram in the 21st Century when molecular diagnostics available? Cheap Universally validated Non specific: one test several organisms Can be combined with rapid protein identification (MALDI-TOF) + nucleic acid tests Pure culture allows elimination of non-pathogens by progressive testing phenotypic characters Required for phenotypic antimicrobial sensitivity testing 24 Polymerase Chain Reaction Double stranded DNA Primer annealing tp single stranded DNA Copying of DNA Template Repeated cycles Synthesis of millions of copies of original (consensus copies) http://www.juliantrubin.com/encyclopedia/biochemistry/pcr.html 25 26 Pitfalls of PCR for Diagnosis Only find targets your primers specifically bind Expensive reagents with short shelf life and specialized technique Single target type per PCR reaction DNA extraction from specimen required Diagnosis of RNA viruses need extra step PCR process makes more of the target Special measures needed to prevent contamination of specimens by PCR products yielding false positives –different rooms for DNA extraction and PCR , environmental treatment with UV lamps 27 Commercial solutions to make PCR workable in busy diagnostic laboratories Automate DNA extraction direct from specimen and PCR and DNA target detection inside a closed pouch so PCR product is never released Dehydrated reagents with long shelf life inside pouch Multiplex assays carrying out 10,15,20 PCRs for different targets in parallel, including reverse transcriptase for RNA targets, in < 2 hours Tailor multiplex panel to most important pathogens typical for specific specimens/syndromes 28 The dried reagents in the FilmArray RP pouch are reconstituted by the addition of 1 ml distilled water to the blue port (lower right of diagram), and the diluted sample is injected into the port shown in red. Kenneth H. Rand et al. J. Clin. Microbiol. 2011; doi:10.1128/JCM.02582-10 29 Panel of 14 PCR targets for meningitis applied to csf samples BACTERIA: Escherichia coli K1 Haemophilus influenzae Listeria monocytogenes Neisseria meningitidis Streptococcus agalactiae Streptococcus pneumoniae VIRUSES: Cytomegalovirus (CMV) Enterovirus (EV) Herpes simplex virus 1 (HSV-1) Herpes simplex virus 2 (HSV-2) Human herpesvirus 6 (HHV-6) Human parechovirus (HPeV) Varicella zoster virus (VZV) 30 Blood culture panel of targets 24 pathogens and 3 antibiotic resistance genes Gram-Positive Bacteria Enterococcus Listeria monocytogenes Staphylococcus Staphylococcus aureus Streptococcus agalactiae Streptococcus pyogenes Streptococcus pneumoniae Gram negative bacteria Acinetobacter baumannii Haemophilus influenzae Neisseria meningitidis Pseudomonas aeruginosa Enterobacter cloacae complex Escherichia coli Klebsiella oxytoca Klebsiella pneumoniae Proteus Serratia marcescens Yeasts Candida glabrata Candida krusei Candida parapsilosis Candida tropicalis Resistance genes mecA - methicillin resistant vanA/B - vancomycin resistant KPC - carbapenem resistant Candida albicans 31 DNA Genome Sequencing Workflow from bacterial colony to DNA extraction to sequence is several days Moore’s law type process in cost per base of sequencing Currently replacing older technology for reference identification and subtyping processes for organisms that grow slowly- mycobacteria (Tuberculosis) Older techniques which are intensive and unwieldy (Salmonella, Neisseria serotyping, Campylobacter speciation etc) Can predict antimicrobial resistance but not guarantee phenotypic sensitivity what u cant use what u can 32 Diagnostic Metagenome sequencing Equivalent to open ended (agnostic) culture of sample, finding all microorganisms present Extract and sequence all DNA/RNA in specimen Bioinformatically exclude host sequence, assemble all other fragments Research technique only at present 33 End of Part 2 True-False Questions 4. MALDI-TOF is a DNA-based bacterial identification system 5. Diagnostic PCR can be directly applied to sterile samples like CSF and blood 6. Virus diagnosis is primarily by culture 34 Part 3: Part 2 True-False Questions 4. MALDI-TOF is a DNA-based bacterial identification system FALSE 4. Diagnostic PCR can be directly applied to sterile samples like CSF and blood TRUE 5. Virus diagnosis is primarily by culture FALSE 35 Diagnosis of urinary tract infection: appropriate specimens Urine Commonest specimen type in Microbiology: mid stream urine (MSU): best for standard urinary tract infection catheter specimen urine(CSU): indwelling catheters > 1 week always infected, treatment not always required early morning urine(EMU): tuberculosis supra-pubic aspirate(SPA), ureteric urine : invasive high value specimens Investigation Urinalysis (stick testing) (Day 1) microscopic examination (Presence of bacteria, inflammatory cells) (Day1 ) Culture (Day2 ) If febrile, rigors Blood cultures 36 Urine specimens Sterile container: ideally refrigerated if not immediately sent to lab Dip stick Dip slide (for remote GPs when refrigeration not possible) 37 Urine Flow Cytometry Replacing Urine Microscopy laser scattering and fluorescence counting host cells + bacteria Requires boric acid addition to sample bottle to preserve cell integrity Boric acid is bacteriostatic 38 Diagnosis of pneumonia: appropriate specimens Respiratory tract specimens sputum, bronchoalveolar lavage Microscopy and culture, immunofluorescence, PCR Day 1 Gram stain – because of passage through mouth bacteria likely to be seen or grown if respiratory tract infection or not PCR preferably done on lower respiratory tract sample to avoid oral contamination Blood culture 20-40% Pneumococcal pneumonia patients have bacteraemia with S. pneumoniae Serology Urinary antigen in acute pneumonia possible on Day 1 Pneumococcal /Legionella polysaccharides excreted in urine Remains positive after antibiotics commenced acute/convalescent antibodies in patients blood, may required weeks to mount detectable immune response 39 Diagnosis of skin/soft tissue sepsis Skin / soft tissue swabs for microscopy and culture on agar Day 1 – Gram stain Day 2- culture Commensal organisms e.g. S.epidermidis likely to be grown with or without significant infection Blood cultures if febrile Biopsy (severe infection) Microscopy, histology and cultures 40 Specimen Pitfalls (Pre-analytical) Unlabelled sample cannot be processed because of misidentification risks May be an invasive sample e.g. csf or biopsy No clinical history Impedes clinical advice/result prioritisation Leaking poorly packaged sample Biohazard for staff 41 Wrong Blood in Tube (WBIT) Cross matching in blood transfusion- potentially fatal event 1% of samples Blood is taken from the wrong patient and is labelled with the intended patient's details (‘miscollected’). Blood is taken from the intended patient, but labelled with another patient's details ( ‘mislabelled’) Outcome False positive or negative diagnosis of infection Unnecessary treatment e.g. TB Delayed diagnosis Failed diagnosis 42 Bedside Barcoding systems used by Phlebotomy in some hospitals in Ireland, not generally medical staff Minimises labelling errors 43 Lecture objectives Describe the importance of correct specimen labelling and packaging by clinicians: (avoiding errors,providing clinical information e.g. foreign travel history reducing infection risks to staff) Name the most common microbiology specimens (urines, swabs, be aware that that MSU is not the generic name for all urine samples) 3. Name less common, more significant samples and understand the difference between sterile and non-sterile sites – blood, csf, BAL etc, 4. Discern the difference between rapid initial tests such as microscopy , Gram staining and culture based tests (in terms of time taken and specificity of result ) 5. Be aware of modern advances in laboratory diagnosis such as Molecular diagnosis , MALDI-TOF 6. Formulate appropriate specimen requests to diagnose specific clinical conditions 44