Diagnostic Medical Microbiology 29 Safar 1430 25 Febuary 2009.

Diagnostic Medical Diagnostic Medical MicrobiologyMicrobiology

29 Safar 143029 Safar 1430

25 Febuary 200925 Febuary 2009

Objectives and Learning Objectives and Learning OutcomesOutcomes

► To acquire basic skills in the identification and isolation To acquire basic skills in the identification and isolation of microbial pathogens.of microbial pathogens.

► Demonstrate understanding of the principles of Demonstrate understanding of the principles of diagnostic medical microbiology and the clinical diagnostic medical microbiology and the clinical correlations.correlations.

► Make observations, understand the fundamental Make observations, understand the fundamental elements of experimental design, generate and analyse elements of experimental design, generate and analyse data on pathogenesis and control of diseases.data on pathogenesis and control of diseases.

► Demonstrate proficiency in collection, interpretation, Demonstrate proficiency in collection, interpretation, and presentation of scientific data in medical and presentation of scientific data in medical microbiology when conducting a research.microbiology when conducting a research.

► Communicate about case studies using appropriate oral Communicate about case studies using appropriate oral and written means.and written means.

Lecture Outline:Lecture Outline:

►Normal Bacterial & Fungal floraNormal Bacterial & Fungal flora►Laboratory Aids in the selection of Laboratory Aids in the selection of

Antimicrobial TherapyAntimicrobial Therapy►Diagnosis of Chlamydial infectionsDiagnosis of Chlamydial infections►Diagnosis of Viral InfectionsDiagnosis of Viral Infections►Diagnosis of HIV and AIDSDiagnosis of HIV and AIDS

Normal Bacterial & Fungal Normal Bacterial & Fungal FloraFlora

► Some organisms are considered pathogens Some organisms are considered pathogens whenever they are found in patients. Eg.whenever they are found in patients. Eg.

► Many infections are caused by organisms that are Many infections are caused by organisms that are permanent members of normal flora. permanent members of normal flora.

► Gram-negative rods are strongly suspect as the Gram-negative rods are strongly suspect as the cause of pneumonia eg. cause of pneumonia eg. Klebsiella pneumoniaeKlebsiella pneumoniae

► In some cases, identification of normal flora is In some cases, identification of normal flora is more warranted, eg. in abdominal abscesses.more warranted, eg. in abdominal abscesses.

► Yeast in small numbers are common flora, not Yeast in small numbers are common flora, not others.others.

► Viruses are usually not part of the normal flora, Viruses are usually not part of the normal flora, but some latent viruses eg. _____________ or live but some latent viruses eg. _____________ or live vaccine viruses _______________ occasionally vaccine viruses _______________ occasionally appear in viral cultures.appear in viral cultures.

Laboratory Aids in the Selection Laboratory Aids in the Selection of Antimicrobial Therapyof Antimicrobial Therapy

► Drug testing is essential in those bacteria commonly Drug testing is essential in those bacteria commonly showing resistance, primarily showing resistance, primarily StaphylococcusStaphylococcus sp., sp., Neisseria gonorrhoeaNeisseria gonorrhoea etc. Testing on fungal and etc. Testing on fungal and protozoan infections is difficult and often protozoan infections is difficult and often unnecessary. Identification of infectious agents should unnecessary. Identification of infectious agents should be attempted as soon as possible, secured before the be attempted as soon as possible, secured before the antimicrobic drug is given.antimicrobic drug is given.

► Choice of drug is based on “informed best guess”.Choice of drug is based on “informed best guess”.► Disk diffusion susceptibilityDisk diffusion susceptibility (or Kirby-Bauer) test – (or Kirby-Bauer) test –

common test, that measures the ability of drugs to common test, that measures the ability of drugs to inhibit the growth of bacteria. The size of zones of inhibit the growth of bacteria. The size of zones of inhibition vary with each different drug.inhibition vary with each different drug.

► Tube dilution testsTube dilution tests give more sensitive and give more sensitive and quantitative results.quantitative results.


► Minimum inhibitory concentrationMinimum inhibitory concentration (MIC) = measures the (MIC) = measures the concentration of an antibiotic necessary to inhibit growth concentration of an antibiotic necessary to inhibit growth of a standardised inoculum under defined conditions. of a standardised inoculum under defined conditions. The end point, or minimum inhibitory concentration is The end point, or minimum inhibitory concentration is the last lowest concentration of drug that gives clear the last lowest concentration of drug that gives clear broth, ie. free from microbial growth. This is very useful broth, ie. free from microbial growth. This is very useful in gauging the dosage regimen necessary for patients.in gauging the dosage regimen necessary for patients.

► In addition, bactericidal effects can be estimated by In addition, bactericidal effects can be estimated by subculturing the clear broth onto antibiotic-free solid subculturing the clear broth onto antibiotic-free solid media. The result, which is a reduction of colony-forming media. The result, which is a reduction of colony-forming units by 99.9% below that of the control, is called the units by 99.9% below that of the control, is called the minimal bactericidal concentrationminimal bactericidal concentration (MBC). (MBC).

► The results of antimicrobic sensitivity tests guide the The results of antimicrobic sensitivity tests guide the physician’s choice of a suitable drug. Continuous physician’s choice of a suitable drug. Continuous observation of patient’s clinical response is imperative observation of patient’s clinical response is imperative once antimicrobial therapy has begun.once antimicrobial therapy has begun.


► Drug toxicity – choose one with high/low Drug toxicity – choose one with high/low selective toxicity for the infectious agent and selective toxicity for the infectious agent and high/low human toxicity.high/low human toxicity.

► Therapeutic index (TI) = the ratio of the dose Therapeutic index (TI) = the ratio of the dose of the drug that is toxic to humans as of the drug that is toxic to humans as compared to its minimum effective compared to its minimum effective (therapeutic) dose:(therapeutic) dose:

► TI = TI = toxic dose ( toxic dose (µg/ml) µg/ml)

effective dose (µg/ml)effective dose (µg/ml)

Hence, TI=10 is ______________than TI=1.2.Hence, TI=10 is ______________than TI=1.2.


► When antimicrobic treatment fails, this may be When antimicrobic treatment fails, this may be due to:due to:

► i) the inability of the drug to diffuse into body i) the inability of the drug to diffuse into body compartment (brain, joints)compartment (brain, joints)

► ii) a few resistant cells that did not appear in ii) a few resistant cells that did not appear in the sensitivity test the sensitivity test in vitroin vitro

► iii) infection caused by more than one iii) infection caused by more than one pathogenpathogen

► Other considerations before administrating Other considerations before administrating drugs: allergy, pregnancy, underlying liver or drugs: allergy, pregnancy, underlying liver or kidney diseases, age, any genetic or metabolic kidney diseases, age, any genetic or metabolic abnormalities, drugs incompatibility, cost.abnormalities, drugs incompatibility, cost.

Diagnosis of ChlamydiaDiagnosis of Chlamydia

► Chlamydia : StructureChlamydia : Structure► Outer cell wall resembles Gram-negative; Outer cell wall resembles Gram-negative;

with relatively high lipid content.with relatively high lipid content.► Cell wall is rigid but no peptidoglycan, hence Cell wall is rigid but no peptidoglycan, hence

no effect on lysozyme. Perhaps it contains no effect on lysozyme. Perhaps it contains tetrapeptide-linked matrix.tetrapeptide-linked matrix.

► Penicillin-binding proteins occur in Penicillin-binding proteins occur in chlamydiae.chlamydiae.

► DNA and RNA are present in elementary and DNA and RNA are present in elementary and reticulate bodies.reticulate bodies.

Diagnosis of Chlamydial Diagnosis of Chlamydial InfectionsInfections

► Although Although Chlamydia spChlamydia sp. are bacteria, they are . are bacteria, they are obligate intracellular parasitesobligate intracellular parasites. Hence, . Hence, procedures are much likely resembled those procedures are much likely resembled those of diagnosing viruses.of diagnosing viruses.

► Specimens are collective from respective Specimens are collective from respective sites of infections, and carefully placed in sites of infections, and carefully placed in transport medium:transport medium:SpeciesSpecies Infected sitesInfected sites

C. C. trachomatistrachomatis

Oculogenital epithelial, Oculogenital epithelial, conjunctival scraping, conjunctival scraping, endometrium scrapingendometrium scraping

C. C. pneumoniaepneumoniae

Nasopharyngeal swabNasopharyngeal swab

CC. psittaci. psittaci Sputum, blood, biopsy material.Sputum, blood, biopsy material.

ChlamydiaeChlamydiae

►Staining propertiesStaining properties::►Giemsa stainsGiemsa stains: Elementary bodies – : Elementary bodies –

purple; larger, noninfective reticulate bodies purple; larger, noninfective reticulate bodies stain – blue and host cytoplasm - blue.stain – blue and host cytoplasm - blue.

►Fully formed, mature intracellular inclusions Fully formed, mature intracellular inclusions of C trachomatis are compact masses near of C trachomatis are compact masses near nucleus (dark purple) in Giemsa stain. nucleus (dark purple) in Giemsa stain.

►Gram staining – negativeGram staining – negative

ChlamydiaChlamydia

► MicroscopyMicroscopy::► Micro-organisms can Micro-organisms can

sometimes be found in the sometimes be found in the cytoplasm of squamous cytoplasm of squamous cells from conjunctival cells from conjunctival scrapings due to scrapings due to ChlamydiaChlamydia psittacipsittaci infection. This infection. This organism forms compact, organism forms compact, oval-shaped grape-like oval-shaped grape-like cluster in the cells’ cluster in the cells’ cytoplasmcytoplasm (yellow arrow) (yellow arrow). . Sometimes dispersed Sometimes dispersed elementary bodies can be elementary bodies can be seen. seen.

Diagnosis of Diagnosis of ChlamydiaChlamydia► Cell cultureCell culture techniques for isolation of chlamydia techniques for isolation of chlamydia

species involves inoculation of the species involves inoculation of the C trachomatisC trachomatis and and C psittaciC psittaci onto cycloheximide-treated McCoy cells onto cycloheximide-treated McCoy cells (antimetabolites that inhibit host cell replication but (antimetabolites that inhibit host cell replication but allows chlamydiae to use available cell nutrient for allows chlamydiae to use available cell nutrient for growth), and growth), and C pneumoniaeC pneumoniae requires pretreated HEp-2 requires pretreated HEp-2 cells. cells.

► ImmunoassayImmunoassay –EIAs are used to detect chlamydial –EIAs are used to detect chlamydial antigens from genital swab. antigens from genital swab.

► Nucleic acid HybridizationNucleic acid Hybridization – commercial kits with – commercial kits with nonradioisotopic probes are available for nonradioisotopic probes are available for C C trachomatistrachomatis 16S RNA sequences. PCR and ligase chain 16S RNA sequences. PCR and ligase chain reaction (LCR) are much more sensitive than culture.reaction (LCR) are much more sensitive than culture.

► SerologySerology: Complement fixation test is widely used to : Complement fixation test is widely used to diagnose psittacosis. The microimmunofluorescence diagnose psittacosis. The microimmunofluorescence method is more sensitive than CF for measuring method is more sensitive than CF for measuring antichlamydial antibodies. Detection of IgM against C antichlamydial antibodies. Detection of IgM against C trachomatis is helpful in infant pneumonitis.trachomatis is helpful in infant pneumonitis.

Diagnosis of Viral InfectionsDiagnosis of Viral Infections

► Isolation of a virus may not establish the Isolation of a virus may not establish the cause of a given disease. Some viruses cause of a given disease. Some viruses persist in human hosts for long periods.persist in human hosts for long periods.

► Care for specimens: refrigerated for up to Care for specimens: refrigerated for up to 24hr (except RSV) or freeze -6024hr (except RSV) or freeze -60°°C or colder.C or colder.

► Virus is present in pharyngeal secretions; fluid Virus is present in pharyngeal secretions; fluid and scrapings of vesicular rashes; and scrapings of vesicular rashes; conjunctival swabs and tears; brain tissue; conjunctival swabs and tears; brain tissue; and CSF; faeces.and CSF; faeces.

► Direct fluorescent antibody is the tests of Direct fluorescent antibody is the tests of choice for diagnosis because they provide choice for diagnosis because they provide answers within a few hours after collection cf. answers within a few hours after collection cf. virus culture.virus culture.

Detection of Viral GrowthDetection of Viral Growth► Lytic or cytopathic viruses replicate in cells and Lytic or cytopathic viruses replicate in cells and

produce alterations in cellular morphology (or cell produce alterations in cellular morphology (or cell death) and the effect can be seen directly by light death) and the effect can be seen directly by light microscopy.microscopy.

► For example, enteroviruses often produce cell For example, enteroviruses often produce cell rounding, pleomorphism and eventual cell death; and rounding, pleomorphism and eventual cell death; and measles and RSV cause fusion of cells to produce measles and RSV cause fusion of cells to produce ____________.____________.

► Other viruses are detectable by their production of Other viruses are detectable by their production of haemagglutinins; haemagglutinins; and by a method called and by a method called interferenceinterference whereby the whereby the virus which produces no cytopathic effect in virus which produces no cytopathic effect in susceptible cell culture, but can be detected by susceptible cell culture, but can be detected by “challenging” the cell culture with a different virus that “challenging” the cell culture with a different virus that normally produces a characteristic cytopathic cell. The normally produces a characteristic cytopathic cell. The second virus fails to infect the cell culture because of second virus fails to infect the cell culture because of the interference by the first virus, which is thus the interference by the first virus, which is thus detected. Eg. Rubella virus. detected. Eg. Rubella virus.

A. Normal monkey kidney cell culture monolayer. B.Enterovirus cytopathic effect in a monkey kidney cell monolayer. Note cell lysis and monolayer destruction. (X 40)

C. Normal human diploid fibroblast cell monolayer. D. Cytomegalovirus cytopathic effect in human diploid cell monolayer. Note rounded, swollen cells in a focal area. (Χ 40)

In vivoIn vivo Isolation Methods Isolation Methods

► Embryonated hen’s egg is still used for the initial Embryonated hen’s egg is still used for the initial isolation and propagation of influenza A virus. isolation and propagation of influenza A virus. The egg is incubated to permit viral replication The egg is incubated to permit viral replication and recognition.and recognition.

► Animal host, commonly the mouse; suckling Animal host, commonly the mouse; suckling mice in the first 48h of life are especially mice in the first 48h of life are especially susceptible to many viruses. Viral replication is susceptible to many viruses. Viral replication is based on development of illness, with signs such based on development of illness, with signs such as paralysis, convulsions, poor feeding or death.as paralysis, convulsions, poor feeding or death.

► Further test to elucidate the nature of infecting Further test to elucidate the nature of infecting virus – by histologic and immunofluorescent virus – by histologic and immunofluorescent examinations of tissues or by antibody examinations of tissues or by antibody detection.detection.

Virus IdentificationVirus Identification

► On isolation, virus can usually be identified by On isolation, virus can usually be identified by its cultural characteristics. Further identification its cultural characteristics. Further identification may require adequate quantities for testing.may require adequate quantities for testing.

► Several diagnostic methods for viral Several diagnostic methods for viral identification are:identification are:

► 1. Antigen detection1. Antigen detection► 2. nucleic acid amplification and detection2. nucleic acid amplification and detection► 3. nucleic acid hybridisation3. nucleic acid hybridisation► 4. measuring immune response to virus 4. measuring immune response to virus

infectioninfection► 5. immune electron microscopy.5. immune electron microscopy.

Diagnostic VirologyDiagnostic Virology

► 1. 1. Antigen detectionAntigen detection: this neutralisation : this neutralisation method works by neutralise virus infectivity by method works by neutralise virus infectivity by mixing it with specific antibody.mixing it with specific antibody.

► Immunofluorescence, enzyme immunoassay Immunofluorescence, enzyme immunoassay (EIA) and latex agglutination are common (EIA) and latex agglutination are common methods.methods.

► Commercial kits are available to detect many Commercial kits are available to detect many viruses inc HSV1 and HSV2, influenza A and B, viruses inc HSV1 and HSV2, influenza A and B, RSV, adenovirus, parainfluenza virus, rotavirus, RSV, adenovirus, parainfluenza virus, rotavirus, cytomegalovirus.cytomegalovirus.

► Advantage: allow detection of viruses that do Advantage: allow detection of viruses that do not readily grow in the cell culture (rotavirus, not readily grow in the cell culture (rotavirus, Hep A), or that grow very slowly (CMV) Hep A), or that grow very slowly (CMV)


► 22. Nucleic Acid Amplification & Detection. Nucleic Acid Amplification & Detection: : commercial assays are available which inc PCR, commercial assays are available which inc PCR, reverse-transcriptase PCR.reverse-transcriptase PCR.

► Detects enteroviruses; and quantifies HIV-1, CMV, Detects enteroviruses; and quantifies HIV-1, CMV, EBV.EBV.

► 3. 3. Nucleic Acid HybridisationNucleic Acid Hybridisation: is a highly sensitive : is a highly sensitive and specific method.and specific method.

► 4. 4. Measuring immune responseMeasuring immune response: cellular immunity : cellular immunity may be assessed by dermal hypersensitivity, may be assessed by dermal hypersensitivity, lymphocyte transformation, cytotoxicity tests. lymphocyte transformation, cytotoxicity tests. Humoral immune responses- IgM appear initially and Humoral immune responses- IgM appear initially and are followed by IgG. IgM disappear in several weeks are followed by IgG. IgM disappear in several weeks whereas IgG persist for many years. The methods whereas IgG persist for many years. The methods used are neutralisation test, complement fixation, used are neutralisation test, complement fixation, haemagglutination inhibition test, and the haemagglutination inhibition test, and the immunofluorescence test. immunofluorescence test.


► 5. 5. Immune Electron Microscopy (EIA)Immune Electron Microscopy (EIA): for : for viruses not detectable by conventional viruses not detectable by conventional techniques.techniques.

► Antigen-antibody complexes or aggregates Antigen-antibody complexes or aggregates formed between virus particles in formed between virus particles in suspension are caused by the presence of suspension are caused by the presence of antibodies in added antiserum and are antibodies in added antiserum and are detected more readily and with greater detected more readily and with greater assurance than individual virus particles.assurance than individual virus particles.

► IEM detects viruses that cause enteritis and IEM detects viruses that cause enteritis and diarrhoea.diarrhoea.

HIV and AIDSHIV and AIDS

► HIV-1 occurs worldwide, while HIV-2 occurs HIV-1 occurs worldwide, while HIV-2 occurs primarily in West Africa and other geographic primarily in West Africa and other geographic areas.areas.

► Blood banks use very sensitive tests to detect Blood banks use very sensitive tests to detect HIV-1 in donated blood.HIV-1 in donated blood.

► Characteristics of HIV:Characteristics of HIV:► Family , RNA,Family , RNA,► Possess viral enzyme reverse transcriptase.Possess viral enzyme reverse transcriptase.► Commonly assayed viral protein is p24. Commonly assayed viral protein is p24.

antibody responses to several HIV gene antibody responses to several HIV gene product: product: envenv, gp160, gp120, gp41, , gp160, gp120, gp41, gaggag and and polpol..

Assays for Anti-HIV Assays for Anti-HIV AntibodiesAntibodies

► A. A. ELISAELISA Enzyme-Linked Immunosorbent Assay Enzyme-Linked Immunosorbent Assay::► Primary screening test for HIV-1 infection.Primary screening test for HIV-1 infection.► HIV-1 antigens immobilised on a solid surface HIV-1 antigens immobilised on a solid surface

(plastic wells, or beads). Add patient’s serum. HIV-1 (plastic wells, or beads). Add patient’s serum. HIV-1 antibodies bound to immobilised antigens are then antibodies bound to immobilised antigens are then detected with an enzyme labeled anti-human IgG detected with an enzyme labeled anti-human IgG and a colorimetric reaction.and a colorimetric reaction.

► Results interpretation: the amount of colour is Results interpretation: the amount of colour is proportionately higher with higher HIV-1 antibodies. proportionately higher with higher HIV-1 antibodies.

► ELISA for HIV-1 is extremely sensitive and specific.ELISA for HIV-1 is extremely sensitive and specific.► What about infants born with HIV-infected mothers?What about infants born with HIV-infected mothers?► ELISA test at 2 months old:ELISA test at 2 months old:► ELISA test at 2 years age:ELISA test at 2 years age:

Principle of Enzyme Principle of Enzyme ImmunoassaysImmunoassays

First Ab Add antigen

Second antibodyfor the Ag, labelled with enzyme

Assays for HIV-1 AntibodiesAssays for HIV-1 Antibodies

►B. B. Western BlotWestern Blot::►To measure specific HIV-1 antibodies To measure specific HIV-1 antibodies

to confirm a positive ELISA result. to confirm a positive ELISA result.

Western BlotWestern Blot► 1. HIV-1 proteins are 1. HIV-1 proteins are

separated by separated by electrophoresis.electrophoresis.

► 2. HIV-1 proteins are 2. HIV-1 proteins are transferred onto transferred onto nitrocellulose strip.nitrocellulose strip.

► 3. the strip is incubated 3. the strip is incubated with patient’s serum. with patient’s serum. The specific HIV-1 The specific HIV-1 antibodies are antibodies are subsequently detected subsequently detected using an enzyme-linked using an enzyme-linked antihuman IgG.antihuman IgG.

► 4. positive colorimetric 4. positive colorimetric reaction forms bands on reaction forms bands on the nitrocellulose paper the nitrocellulose paper corresponding to p24, corresponding to p24, gp41 and pg120/160.gp41 and pg120/160.

B. Western Blot: ResultsB. Western Blot: Results

► i) Positive test : any 2 bands corresponding i) Positive test : any 2 bands corresponding to the proteins.to the proteins.

► ii) no bands : negative resultii) no bands : negative result► iii) bands that do not meet the criterion for a iii) bands that do not meet the criterion for a

positive test is an indeterminate results.positive test is an indeterminate results.► False-positive and false-negative results are False-positive and false-negative results are

relatively uncommon.relatively uncommon.► Repeat positive ELISAs and indeterminate Repeat positive ELISAs and indeterminate

Western blots.Western blots.► Infant born to an HIV-1-infected mother?Infant born to an HIV-1-infected mother?

Assays to Detect HIV Assays to Detect HIV InfectionInfection

► A. A. Detection of p24 AntigenDetection of p24 Antigen::► ELISA to detect p24 antigen. p24 antigen is detectable ELISA to detect p24 antigen. p24 antigen is detectable

during the acute viraemic stage and in the late stages during the acute viraemic stage and in the late stages of AIDS.of AIDS.

► A very small proportion of asymptomatic HIV-1 infected A very small proportion of asymptomatic HIV-1 infected persons are p24 antigen +.persons are p24 antigen +.

► B. B. Detection of HIV-1 RNA: Detection of HIV-1 RNA: commercial assays inc PCR. commercial assays inc PCR. NASBA, bDNA.NASBA, bDNA.

► C. C. HIV-1 Proviral DNAHIV-1 Proviral DNA: DNA extracted from : DNA extracted from mononuclear cells obtained from anticoagulated mononuclear cells obtained from anticoagulated peripheral blood. Perform PCR. Useful for infants.peripheral blood. Perform PCR. Useful for infants.

► D. D. HIV-1 CultureHIV-1 Culture: peripheral blood mononuclear cells : peripheral blood mononuclear cells from a potentially infected patient are cocultured with from a potentially infected patient are cocultured with uninfected cells stimulated with phyto haemagglutinin uninfected cells stimulated with phyto haemagglutinin and interleukin-2. Cultures are looked for: and interleukin-2. Cultures are looked for: multinucleated giant cells, HIV-1 rev transcrip, p24 Ag. multinucleated giant cells, HIV-1 rev transcrip, p24 Ag. Time-consuming and expensive.Time-consuming and expensive.

Objectives and Learning Objectives and Learning OutcomesOutcomes

► To acquire basic skills in the identification and isolation To acquire basic skills in the identification and isolation of microbial pathogens.of microbial pathogens.

► Demonstrate understanding of the principles of Demonstrate understanding of the principles of diagnostic medical microbiology and the clinical diagnostic medical microbiology and the clinical correlations.correlations.

► Make observations, understand the fundamental Make observations, understand the fundamental elements of experimental design, generate and analyse elements of experimental design, generate and analyse data on pathogenesis and control of diseases.data on pathogenesis and control of diseases.

► Demonstrate proficiency in collection, interpretation, Demonstrate proficiency in collection, interpretation, and presentation of scientific data in medical and presentation of scientific data in medical microbiology when conducting a research.microbiology when conducting a research.

► Communicate about case studies using appropriate oral Communicate about case studies using appropriate oral and written means.and written means.

Diagnostic Medical Microbiology 29 Safar 1430 25 Febuary 2009.

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Transcript of Diagnostic Medical Microbiology 29 Safar 1430 25 Febuary 2009.