laboratory diagnosis of infectious diseases. objectives know available diagnostic technologies for...

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  • OBJECTIVES Know available diagnostic technologies for ID Understand specific specimen for specific diagnostic test Understand procedure for specimen collection
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  • DIAGNOSTIC TECHNOLOGIES Gram stain/Microscopy Cultures. Three major culture media Enrichment: chocolate and sheep blood Selective: Thayer-Martin Differential: MacConkey-ability to ferment lactose
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  • DIAGNOSTIC TECHNOLOGIES Antigen Detection Assays Most has poor sensitivity and specificity 57% sensitivity and 98% specificity for pneumococcal pneumonia Conc. Urine EIA for Legionella pneumophila serogroup 1 has 89% sensitivity and 100% specificity Immunochromatographic assay has better sensitivity and are faster
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  • DIAGNOSTIC TECHNOLOGIES Immunoserology Hemagglutination EIA Latex agglutination Compliment fixation Immunoflorecent
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  • LIMITATIONS OF CONVENTIONAL CLINICAL MICROBIOLOGY Culture Labor intensive Need for special media Prolonged period of time to culture Some organisms are uncultivable on artificial media Potential health hazards Antigen Detection Negative tests require confirmation Effected by poor specimen collection Low microbe burden Serology Unhelpful during early stage of infection Not quite useful in immunocompromised patients
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  • MOLECULAR DIAGNOSTICS Most widely used is PCR High sensitivity High specificity Diversity Nucleic acid probes Do not amplify DNA
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  • MOLECULAR DIAGNOSTICS Polymerase Chain Reaction Specific PCR: Uses primers to known DNA targets. So far 31 clinical bacterial gene sequence are known and 38 in progress Use when conventional diagnostics are inadequate, time consuming, difficult and hazardous Broad range PCR: uses complementary primers to conserved regions shared by a given taxonomic group Used in cases of B. henselae and Mycobacterium spp
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  • MOLECULAR DIAGNOSTICS Multiplex PCR Uses single clinical specimen to investigate several potential pathogens simultaneously Encephalitis/meningitis panel: HSV,VZV, CMV HHV-6, EBV, Enteroviruses Real-time PCR Utilizes a fluorescent labeled probe Requires small volumes thus takes 30-60 minutes to complete
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  • Leading uses for nucleic acid based tests Nonculturable agents Human papilloma virus Hepatitis B virus Fastidious, slow-growing agents Mycobacterium tuberculosis Legionella pneumophilia Highly infectious agents that are dangerous to culture Francisella tularensis Brucella species Coccidioidis immitis
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  • Leading uses for nucleic acid based tests In situ detection of infectious agents Helicobacter pylori Toxoplasma gondii Agents present in low numbers HIV in antibody negative patients CMV in transplanted organs Organisms present in small volume specimens Intra-ocular fluid Forensic samples
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  • Leading uses for nucleic acid based tests Differentiation of antigenically similar agents May be important for detecting specific virus genotypes associated with human cancers (Papilloma viruses) Antiviral drug susceptibility testing May be important in helping to decide anti-viral therapy to use in HIV infections Non-viable organisms Organisms tied up in immune complexes
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  • Leading uses for nucleic acid based tests Molecular epidemiology To identify point sources for hospital and community-based outbreaks To predict virulence Culture confirmation
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  • OTHER USES OF MOLECULAR DIAGNOSTICS Viral load monitoring Viral genotyping Bacterial resistance detection Bacterial genotyping
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  • LIMITATION OF PCR TECHNOLOGIES Cost False positives caused by amplification of contaminants Only sample from normally sterile sites should be considered for broad-range PCR Specimen is required to be refrigerated or stored in alcohol before processing
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  • LIMITATION OF PCR TECHNOLOGIES Specimen should be frozen until amplification No antimicrobial sensitivity is available Needs the clinician to name the suspect
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  • RAPID DIAGNOSTIC TESTS High sensitivity and specificity High negative and positive predictive values High accuracy compared to gold standard Simple to perform Rapid turn around time Cost effective
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  • PathogensMechanismSensitivity (%) Specificity (%) Positive Predictive Value (%) Negative Predictive Value (%) Time to perform test Group A Streptococcus Detects group A staphylococcal carbohydrate antigen by immunoassay 89-94 (Compared to culture) 95-9955-8990-975 minutes Helicobacter pyloriDetects immunoglobulin G antibodies specific to H. pylori 85-90 (Compared to biopsy) 80-8985795-10 minutes Borrelia burgdorferiDetects antibodies to B. burgdorferi using recombinant antigen 72 (Compared to ELISA) 97Not available 20 minutes
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  • Collection and Processing of Clinical Specimen Diagnostic Technologies Culture Antigen detection Serology Molecular diagnostics Rapid Diagnostic Test CLIA-Waived tests Other rapid non-CLIA waived tests
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  • COLLECTION AND PROCESSING OF CLINICAL SPECIMEN Most important aspect of laboratory medicine Insufficient quantity Contamination Improper transport media Delay in transportation Inappropriate storage
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  • COLLECTION AND PROCESSING OF CLINICAL SPECIMEN Collecting Blood Clean with 70% ethyl alcohol Disinfect with 10% povidone-iodine Allow to dry for at least 1 minute No wiping! Clean the rubber stopper of the bottle Use alcohol for Bactec bottle to prevent cracking
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  • COLLECTION AND PROCESSING OF CLINICAL SPECIMEN Collect enough blood 1-2ml in neonate 2-3ml in infants 3-5ml in children 10-20ml in adolescent Rapid inoculation A 3 hour delay result in 25% reduction in recovery of S. pneumoniae Paisley JW, Lauer BA. Pediatric blood cultures. Clin Lab Med 1994; 14: 17 Roback MG, Tsai AK, Hanson KL. Delayed incubation of blood culture bottles: Effect on recovery rate of S. pneumoniae. Pediatr Emerg Care 1994; 10: 268
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  • COLLECTION AND PROCESSING OF CLINICAL SPECIMEN Collecting urine Clean-voided midstream urine Use of urine bag Catheterized specimen/ Suprapubic aspiration Collecting CSF CSF is hypotonic Refrigeration can render fastidious bacteria non-viable Cell count decreases by 32% after 1 hour and 50% after 2 hours Steele RW, Mormer DJ, OBrien MD, et al. Leukocyte survival in cerebrospinal fluid. J Clin Microbiol 1986; 23: 965
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  • COLLECTION AND PROCESSING OF CLINICAL SPECIMEN Insufficient quantity/quality Small quantity for optimal analysis Poor specimen e.g. eye cultures for chlamydiae should have enough cellular element Contamination During collection During transport Contamination in the lab
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  • COLLECTION AND PROCESSING OF CLINICAL SPECIMEN Improper transport media Prevent drying Maintain optimal physiochemical environment Prevent oxidation and destruction of enzymes Provide adequate nutrients Three major culture media Enrichment: chocolate and sheep blood Selective: Thayer-Martin Differential: MacConkey-ability to ferment lactose
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  • COLLECTION AND PROCESSING OF CLINICAL SPECIMEN Delay in transportation Holding conditions are specimen or pathogen specific Urine: 2 C to 8C Inoculated blood: 35 C to 37C
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  • SPECIFIC EXAMPLES Specimen for isolation of N. gonorrhoeae should be inoculated into a specific media, transported within 30 minutes of collection, incubated at 35-37C in 5-10% co 2 Stool for ova & parasite should be placed in preservatives CSF is held in room temperature and never refrigerated Stool for C. difficle must be refrigerated or frozen
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  • SUMMARY If you are not sure of how to collect a specimen call microbiology before collection If you are unsure what to send, call ID before collection If you are not sure of specific clinical syndrome, save a sample
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  • You are asked to attend the delivery of a term infant because the baby is small for gestational age, and prenatal ultrasonography revealed periventricular cerebral calcifications. The infants birth weight is 2,000 g. On physical examination, you note hepatosplenomegaly and a petechial rash on the face and trunk. Of the following, the BEST laboratory test for diagnosing the cause of these findings is A.Nasopharyngeal culture for herpes simplex B.Rapid plasma reagin for syphilis C.Serum immunoglobulin (Ig) G titer for rubella D.Serum IgM titer for toxoplasmosis E.Urine culture for cytomegalovirus
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  • A 12-year-old girl comes to your office complaining of headache, malaise, fever to 101 F (38.3 C), rhinorrhea, and a sore throat for the past 3 to 4 days. Most recently, she developed hoarseness and cough. On physical examination, you note crackles and wheezes throughout the lung fields. Chest radiography demonstrates interstitial infiltrates in the lower lung fields bilaterally. Of the following, the BEST test to order to confirm the patients diagnosis is: A.Direct fluorescent antibody for Bordetella pertussis B.Enzyme immunoassay for respiratory syncytial virus C.Serology for Mycoplasma pneumoniae D.Throat culture for g

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