Principle of Lab Diagnosis of Infectious Disease

PRINCIPLE OF LAB DIAGNOSIS OF INFECTIOUS DISEASE TEST CYCLE PREANALYTICAL ( sampling, labelling, transport, storage, specimen clinical information) ANALYTICAL (macroscopic evaluation, microscopy, culture identification, interpretation, antibiograme, …) POST ANALYTICAL ( final report to physician) INSTRUCTIONS FOR COLLECTION AND TRANSPORT OF SPECIMENS FOR CULTURE Type of Culture Specimen Minimal Volume Container Other Considerations Blood Whole blood 10 mL ,5 mL in each of 2 bottles for adults and infants; less for neonates 2 bottles one with dextrose phosphate, trypticase soy, or another appropriate broth and the other with thioglycollate or another broth containing reducing agents appropriate for isolation of obligate anaerobes Do not allow air bubbles to get into anaerobic broth bottles. Nose Swab from nares 1 swab Sterile culturette or similar transport system containing holding medium Swabs made of calcium alginate may be used. Throat Swab of post pharynx, ulcerations, or areas of suspected purulence 1 swab Sterile culturette or similar transport system containing holding medium When Neisseria gonorrhoeae or Corynebacterium diphtheriae is suspected, a special culture request is recommended. Sputum Fresh sputum (not saliva) 2 mL sputum collection system or similar sterile container with screw cap Examination of Gram’s stain, with number of epithelial cells and polymorphonuclear leukocytes (PMNs) noted. Bronchial aspirates Transtracheal or bronchial aspirate, bronchoscopy specimen, 1 mL of aspirate or brush in transport medium Sterile aspirate or bronchoscopy tube, bronchoscopy brush in a separate sterile container Special precautions may be required, depending on diagnostic considerations (e.g., Pneumocystis Stool Rectal swab or (preferably) fresh, randomly collected stool 1 g of stool or 2 rectal swabs Plastic-coated cardboard cup or plastic cup with tight-fitting lid or Other leak proof containers. If Vibrio spp. are suspected, the laboratory must be notified, and appropriate collection/transport methods should be used. Urine Clean-voided urine specimen or urine collected by catheter 0.5 mL Sterile, leak-proof container with screw cap or special urine transfer tube See below* Urogenital secretions Vaginal or urethral secretions, cervical swabs, uterine fluid, prostatic fluid, etc. 1 swab or 0.5 mL of fluid Vaginal and rectal swabs transported in Amies transport medium; direct inoculation preferred for Neisseria gonorrhoeae For detection of multiple organisms (e.g., group B Streptococcus, Trichomonas, Chlamydia, or Candida spp.), 1 swab per test should be obtained. Cerebrospin al fluid (lumbar puncture) Spinal fluid 1 mL for routine cultures; ≥5 mL for Mycobacterium Sterile tube with tightfitting cap Do not refrigerate; transfer to laboratory as soon as possible. Body fluids Aseptically aspirated body fluids 1 mL for routine cultures Sterile tube with tightfitting cap. For some body fluids (e.g., peritoneal lavage), increased volumes are helpful for isolation of small numbers of bacteria. Wounds Purulent material or abscess contents obtained from wound 2 swabs or 0.5 mL of aspirated pus sterile tube with tightfitting screw cap. For simultaneous anaerobic cultures, send specimen in anaerobic transport device or closed syringe. should be collected in a syringe (rather than with a swab) to provide an adequate sample volume and an anaerobic environment. Fungi Specimen types listed above may be used. When urine or sputum is cultured for fungi, a first morning specimen usually is preferred. 1 mL or as specified above for individual listing of specimens. Sterile, leak-proof container with tight-fitting cap Specimen should be transported to microbiology laboratory within 1 h of collection. Mycobacteri um (acidfast bacilli) Sputum, tissue, urine, body fluids 10 mL of fluid or small piece of tissue. Swabs should not be used. Sterile container with tight-fitting cap Multiple cultures from the same patient are encouraged. Culturing in liquid media shortens time to detection. Anaerobic organisms Aspirated specimens from abscesses or body fluids 1 mL of aspirated fluid, 1 g of tissue, or 2 swabs An appropriate anaerobic transport device is required Specimens cultured for obligate anaerobes should be cultured for facultative bacteria as well Viruses Respiratory secretions, wash aspirates from respiratory tract, nasal swabs, blood samples (including buffy coats), vaginal and rectal swabs, swab specimens from suspicious skin lesions, stool samples (in some cases) 1 mL of fluid, 1 swab, or 1 g of stool in each appropriate transport medium Fluid or stool samples in sterile containers or swab samples in viral culturette devices (kept on ice but not frozen). Plasma samples and buffy coats in sterile collection tubes at 4–8°C. If specimens are to be shipped or kept for a long time, freezing at −80°C. Most samples for culture are transported in holding medium containing antibiotics to prevent bacterial overgrowth and viral inactivation *(1) Clean-voided specimens, mid void specimens, and Foley or indwelling catheter specimens that yield 50,000 organisms/mL and from which no more than three species are isolated should have organisms identified. Neither indwelling catheter tips nor urine from the bag of a catheterized patient should be cultured. (2) Straight-catheterized, bladder-tap, and similar urine specimens should undergo a complete workup (identification and susceptibility testing) for all potentially pathogenic organisms regardless of colony count. (3) Certain clinical problems (e.g., acute dysuria in women) may warrant identification and susceptibility testing of isolates present at concentrations of <50,000 organisms/ mL. FACTORS LIMITING USEFULNESS OF INVESTIGATIONS: Wrong sample  Delay in transport/ inappropriate storage  Overgrowth by contaminants  Insufficient sample/sampling error  Patient has received antibiotics LABORATORY INVESTIGATION OF MICROBIAL INFECTIONS Examining specimens to detect, isolate and identify pathogens or their products using: 1- Microscopy 2- Culture techniques 3- Biochemical reactions 4- Serological identification 5- Molecular biology techniques 6- Bacteriophage typing EXAMINATION OF SPECIMENS FOR MICROORGANISMS MICROSCOPY Microorganisms can be examined microscopically for their motility, morphology and staining reactions: o Examining pathogen in wet preparations to examine motility of bacteria  capsule  Specimens for fungi. o Staining smears Gram staining  Ziehl-Neelsen technique for AFB  Auramine-phenol technique for AFB  Methylene blue technique  Wayson’s bipolar staining of bacteria  Albert staining of volutin granules  Giemsa technique  Acridine orange fluorochrome staining  Methenamine silver stain or calcofluor white for fungi CULTURE TECHNIQUES o   o         • • • Culture media are used for: Isolation and identification of pathogenic organisms Antimicrobial sensitivity tests Types of culture media: for bacterial isolation & identification Basic Enriched Selective Enrichment Indicator/differential Transport Identification Based on consistencySolid Semisolid Fluid  o o o       Culturing of anaerobesEssential for strict anaerobes such as Clostridium species, bacteroides species, and anaerobic streptococci. Helps to differentiate pathogen and isolate facultative anaerobes from specimens containing commensals. Techniques for obtaining anaerobic conditionsCommercially produced sachets containing oxygen removing chemicals- do not produce hydrogen so not require a catalyst, i.e. are non gas generating system. e.g. of system using sachets- Anaerocult system; AneroGen system Copper coated steel wool to remove oxygen Reducing agents in culture media-e.g.- thyoglycollate broth(reducing agent- sodium thyoglycollate;indicatormethylene blue/ resazurine),cooked meat medium A simple way of providing anaerobic conditions in litmus milk medium, peptone water media, and broths by using an iron strip or iron nail to remove the oxygen. The strip or nail is flame sterilised and while still hot it is dropped into the medium. When the medium has cooled, can be inoculated. Anaerobic jars Culturing in carbon dioxide Once bacteria are isolated, characteristics that are readily detectable after growth on agar media (colony size, colour, haemolytic reactions, odour, microscopic appearance) may suggest a species, but definitive identification requires additional tests. For virus isolation- monolayer of cultured mammalian cells sensitive to infection with the suspected virus Required— I. when the presence of serum antibody is not a criterion for active infection, II. when serologic diagnosis is not practical, or III. when immunoassays have inadequate sensitivity. Virus may be detected by-- 1.direct observation of the cultured cells for cytopathic effects or 2. immunofluorescent detection of viral antigens after incubation. For fungal isolation- Sabouraud dextrose agar (sabouraud glucose neopeptone agar), Czapek- Dox medium, corn meal agar and other media. BIOCHEMICAL REACTIONS COMMON TESTS TO IDENTIFY BACTERIAL ISOLATE Use of substrates and sugars to identify pathogens: a- Sugar fermentation:  Organisms ferment sugar with production of acid only  Organisms ferment sugar with production of acid and gas  Organisms do not ferment sugar b- Production of indole:  Depends on production of indole from amino acid tryptophan   Indole is detected by addition of Kovac’s reagent (pdimethylaminobenzaldehyde) Appearance of red ring on the surface c- H2S production:  Depends on production H2S from protein or polypeptides  Detection by using a strip of filter paper containing lead acetate d- Methyl red reaction (MR):  Fermentation of glucose with production of huge amount of acid  Lowering pH is detected by methyl red indicator e- Voges proskaur’s reaction (VP):  Production of acetyl methyl carbinol from glucose fermentation  Acetyl methyl carbinol is detected by addition KOH  Color of medium turns pink (positive) f- Oxidase test:  Some bacteria produce Cytochrome Oxidase enzyme  Detection by adding few drops of colorless oxidase reagent (tetra methyl p-phenylene diamine hydrochloride) Colonies turn deep purple in color (positive)  g- Catalase test:  Some bacteria produce catalase enzyme  Addition of H2O2 lead to production of brisk effervescence (O2 production) h- Coagulase test:    Some bacteria produce coagulase enzyme Coagulase enzyme converts fibrinogen to fibrin (plasma clot) Detected by slide or test tube method i- Urease test:    Some bacteria produce urease enzyme Urease enzyme hydrolyze urea with production of NH3 Alkalinity of media and change color of indicator from yellow to pink J- Citrate utilization test:  Simon's citrate medium has citrate as sole source of carbon  Change in color to bright blue from green or growth. CITRATE TEST SEROLOGICAL IDENTIFICATION A- Direct serological tests:  Identification of unknown organism  Detection of microbial antigens by using specific known antibodies  Serogrouping and serotyping of isolated organism B- Indirect serological tests:  Detection of specific and non specific antibodies  (IgM & IgG) by using antigens or organisms Serological Assays          Detection antibody in the patient’s serum A current infection should be IgM positive A 4-fold or greater rise on antibody titre The convalescent sample is usually taken 1014 days after the acute sample. Major drawbacks A single IgG antibody titre is difficult to interpret, of course. In certain diseases, a single titre of sufficient magnitude can be used as presumptive evidence Some exceptions IMMUNOSEROLOGICAL TESTS      Hemagglutination EIA Latex Agglutination Complement fixation Immuno flourescent antibody tests 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 MOLECULAR BIOLOGY TECHNIQUES A- Genetic probes (DNA or RNA probes): Detection of a segment of DNA sequence (gene) in unknown organism using a labelled probe  Probe: consists of specific short sequence of labelled single-stranded DNA or RNA that form strong covalently bonded hybrid with specific complementary strand of nucleic acid of organism in question B- Polymerase chain reaction (PCR): Amplification of a short sequence of target DNA or RNA. Then It is detected by a labelled probe. Different versions of PCR :1. Simplex PCR 2. Nested PCR 3. Multiplex PCR 4. Real-time (quantitative ) PCR C- Plasmid profile analysis: Isolation of plasmids from bacteria and determination of their size and number compared with standard strains by agarose gel electrophoresis REAL TIME PCR FOR DIAGNOSIS OF INFECTIOUS DISEASE o Detect PCR product during synthesis o Requires fluorescence-based detection and specialized detection instrumentation o Advantages  Less time for results  Improved analytical sensitivity  Broad applicability (target characterization, load determination etc) OTHER USES OF MOLECULAR DIAGNOSTICS  Viral load monitoring  Viral genotyping  Bacterial resistance detection  Bacterial genotyping BACTERIOPHAGE TYPING Bacteriophages are viruses that parasitize bacterial cell PRACTICAL APPLICATIONS USING PHAGES  Phages are important as a research tools  Phages are used as vectors in DNA recombinant technology  Phage typing of bacteria is important in tracing source of infection for epidemiologic purposes ANTIMICROBIAL SUSCEPTIBILITY TESTING Antimicrobial Susceptibility testing are of two types Dilution tests  Diffusion tests 1.DILUTION TEST Serial dilutions of the antibiotic are prepared and inoculated with a standardized number of organisms and incubated for a prescribed time.  Employed when he therapeutics dose is to be regulated accurately, for tests on slow growing bacteria, and when small degrees of resistance are to be demonstrated  Done by two methods1. Tube dilution method 2. Agar dilution method Different concentrations of Tetracycline in Nutrient broth: Conc. in mcg/ml （microgram） Tetracycline, generally considered a bacteriostatic antibiotic, for this bacterium, has an MIC of 1.6 mcg/ml 2.DIFFUSION METHOD  The drug is allowed to diffuse through the solid medium so that a gradient is established, conc. Highest at the site of application of drug and decreasing with distance. The test bacterium is seeded on the medium and sensitivity is determined by inhibition of growth.  where a large number of isolates are tested for susceptibility to numerous antibiotics.  Disc diffusion( KIRBY- BAUER METHOD) being the common method uses a paper disk impregnated with a fixed concentration of an antibiotic.  The diameter of the inhibition zone is a function of the amount of drug in the disk and susceptibility of the microorganism.  This test must be rigorously standardized since zone size is also dependent on:  Diffusibility of drug  Disc concentration  Medium composition,  Inoculum size,  Temperature of incubation,  Presence of inhibitory or stimulatory substances  Time of incubation  Excess moisture  pH and Thickness of the medium.  Zone diameter can be correlated with susceptibility as measured by the dilution method.  Further correlations using zone diameter allow the designation of an organism as "susceptible", "intermediate", or "resistant" to concentrations of an antibiotic which can be attained in the blood or other body fluids of patients requiring chemotherapy. E-TEST (EPSILOMETER TEST) Recent modification of agar diffusion sensitivity test employing a quantitative diffusion gradient An absorbent strip impregnated with known gradient of drug concentration along its length placed on agar plate seeded with the test bacterium, the antibiotic diffuse in the medium and the MIC is noted. Minimum inhibitory concentration (MIC) : Lowest concentration of the drug that inhibits the growth. Minimum bactericidal concentration (MBC) :  Lowest concentration of drug that kills the bacterium. Can be estimated by sub culturing from the broth tubes that show no growth on to suitable solid media. SUSCEPTIBILITY TESTING OF FUNGAL AGENTS Several systems have been approved for antifungal susceptibility testing. These methods, which determine the minimal fungicidal concentration (MFC), are similar to the broth microdilution methods used to determine the MIC for bacteria. The E-test method is approved for testing the susceptibility of yeasts to fluconazole, itraconazole, and flucytosine, and disk diffusion can be used to test the susceptibility of Candida species to fluconazole and voriconazole. Methods for determining the MFC against fungal agents such as Aspergillus species are technically difficult, and most clinical laboratories refer requests for such testing to reference laboratories. AUTOMATED SYSTEMS Basic principles:       Turbidity as growth indicator. Colorimetric determination of growth. Radioactive method of detection. Bioluminescence assays. Reflectance measurement. Electrical impedance. Measurement of ions in system. AUTOMATED SYSTEMS  ABCS (automated blood culture system)suppliers:BacT/Alert (BioMerieux), BACTEC FX (BD), Versa TREK  Rapid identification system– api 20 E strips  Rapid identification system with AST Vitek 2 BD Phoenix Microscan Walkaway (Dade Behring) 1. 2. 3.  MALDI TOF (Matrix assisted LASER Desorption IonisationTime of Flight Mass spectrometry)