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Principle laboratory diagnosis of infectious diseases

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dr.Ihsan alsaimary

The document discusses laboratory diagnosis of infectious diseases. It covers proper selection, collection and transport of clinical specimens; laboratory tests including microscopy, culture techniques, and biochemical reactions; and specimens from different parts of the body. Key points are the importance of collecting specimens properly before antimicrobial treatment and transporting them quickly to preserve microorganisms. A quality laboratory uses quality assurance and quality control to accurately diagnose infectious agents.

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Principle Laboratory Diagnosis of Infectious Diseases
Outline Rejection of specimens 04 CLINICAL SPECIMENS 05 Laboratory Investigation of Microbial infections 06 COMMUNICATION BETWEEN PHYSICIAN AND LABORATORY 01 specimens 02 QUALITY ASSURANCE AND QUALITY CONTROL 03
COMMUNICATION BETWEEN PHYSICIAN AND LABORATORY • The techniques used to characterize infectious depending on the clinical syndrome and the type of infectious agent. • Because no single test will permit isolation or characterization of all potential pathogens, clinical information is much more important for diagnostic microbiology than it is for clinical chemistry or hematology.
Diagnosis of infectious diseases • The proper diagnosis of an infectious disease requires : • (1) patient history. • (2) physical examination of the patient. • (3) carefully evaluating the patient’s signs and symptoms. • (4) conducting appropriate laboratory tests and other methods. • (5) proper selection, collection, and transport of appropriate clinical specimens.
Guidelines for the collection and transportation of specimens must emphasize two important aspects • I-Collection of the specimen before the administration of antimicrobial agents. • II-Prevention of contamination of the specimen with externally present organisms or normal flora of the body.
The Three Components of Specimen Quality Component Explanation Proper selection of the specimen The specimen must be the appropriate type to diagnose the infectious disease suspected by the clinician. Proper collection of the specimen Proper collection eliminates or minimizes contamination of the specimen with microflora. Sometimes special collection devices are required. Proper transport of the specimen The specimen Transport let viable and/or preserves agent morphology (e.g., rapid transport, sometimes by packing the container in ice or by using a preservative)
PROPER SELECTION, COLLECTION AND TRANSPORT OF CLINICAL SPECIMENS 1-The specimen must be properly selected. Collect the appropriate type of specimen for diagnosis of the infectious disease. 2-The specimen must be properly and carefully collected. 1-From a site of pathogen is most likely to be found. 2-Before antimicrobial therapy has begun 3-The acute stage is the best 4-Avoid harming the patient and causing discomfort. 5-Sufficient quantity. 6-All specimens should be placed or collected into a sterile container 3-Specimens must be properly transported to the lab Specimens should be protected from heat and cold to ensure represent the number and types of organisms present at the time of collection.
QUALITY ASSURANCE AND QUALITY CONTROL IN THE CLINICAL MICROBIOLOGY LABORATORY •A-Quality Assurance (QA) •B-Quality Control (QC)
Quality Assurance (QA) • It is continuously identify, monitor, evaluate, and improve the reliability
Quality Control (QC) • Is designed to monitor the accuracy, reliability, and reproducibility of all tests performed within the laboratory and to identify and correct any problems that exist. • Components of a CML’s QC program include the following: • 1-Standard operating 2-Test verification. procedures manual. 3-Test methods and procedures. The procedures written for every aspect of CML work. use methods for most accurate The QC program must monitor, document, and evaluate all aspects of every test procedure that is performed in the CML.
• 4-Media, reagents, staining solutions, antisera. • 5-Equipment and instruments. • 6-Records and reports. 7-Proficiency testing. Avoid expiration dates maintained and monitored of equipment for performance. QC measures and documented positive and negative controls used for every test procedure
Criteria for rejection of specimens • i-Missing or inadequate identification. • ii-Insufficient quantity. • iii-collected in an inappropriate container. • iv-Contamination suspected. • v-Inappropriate transport or storage. • vi-Unknown time delay. • vii-Haemolysed blood sample.
CLINICAL SPECIMENS FROM VARIOUS ANATOMICAL SITES AND ORGAN SYSTEMS • Circulatory System • Skin, Abscess, and Wound Specimens • Eyes and Ears • Respiratory System • Central Nervous System • Urinary Tract • Genital Tract • Oral Cavity • Gastrointestinal Tract • Body Fluids
Circulatory System •Blood • Blood is usually sterile. • Bacteremia:presence of bacteria in the bloodstream. • Septicemia condition: bacteria product or any toxic substance in blood. • The severe types of septicemia are caused by Gram-negative bacilli that release endotoxin from their cell walls, it can induce fever and septic shock, which can be fatal.
phlebotomy
Principle laboratory diagnosis of infectious diseases
Principle laboratory diagnosis of infectious diseases
Lymphatic System • The lymphatic system consists of lymphatic vessels, lymphoid tissue (including lymph nodes, tonsils, thymus, and spleen), and lymph (the liquid that circulates through the lymphatic system),contains many lymphocytes. • specimens :culturing lymphnode and/or by blood culture, (if the pathogens have entered the bloodstream). • Lymphadenopathy: Diseased lymph nodes.
Skin, Abscess, and Wound Specimens • skin is a type of host defense mechanism, serving as a physical barrier. • Sebaceous glands: Glands in the dermis that usually open into hair follicles and secrete an oily substance known as sebum. Folliculitis: Inflammation of a hair follicle Sty (or stye): Inflammation of a sebaceous gland that opens into a follicle of an eyelash. Furuncle: A localized pyogenic (pus-producing) infection of the skin, usually resulting from folliculitis; also known as a boil.
Obtaining specimens from the skin: • 1-For pustules : • The covering removed using a sterile needle, then fluid and basal cells collected. • 2-For petechiae : • specimen is collected by vigorously scraping the outer margin of the lesion. • 3-Collecting abscess specimens: • the abscess contents are aspirated using a needle and syringe. • 4-Collecting wound specimens: • specimen should be an aspirate, or a sample taken from the advancing margin of the lesion.
Eyes and Ears •Eyes • infectious diseases of the eye include the following: • Conjunctivitis: An infection or inflammation of the conjunctiva. • Keratitis: An infection or inflammation of the cornea.
• Keratoconjunctivitis: An infection that involves both the cornea and conjunctiva. • specimen as conjunctival swab, corneal scraping, or aspirate. • Using separate sterile swabs, sample the conjunctiva of both eyes, even if only one eye is infected.
•Ears • There are three pathways for pathogens to enter the ear: • (1) Eustachian tube, from the throat and nasopharynx. • (2) from the external ear. • (3) via blood or lymph. • otitis media Infection of the middle ear • otitis externa infection of the outer ear canal. • specimen collected by swab of external ear canal, fluid aspirated from the middle ear,
Respiratory System Specimen: • A. Nasal swabs • B. Nasopharyngeal / Pernasal swabs • C. Nasopharyngeal aspirates • D. Nasal washings • E. Throat swabs • F. Lung aspirations • G. Open lung biopsy
Central Nervous System • The nervous system is composed of the central nervous system (CNS) and the peripheral nervous system. • It is sterilized but agent enter through trauma, blood and lymph or along the peripheral nerves. • Glucose level indicator for infection. • Encephalitis: Inflammation of the brain. • Encephalomyelitis: Inflammation of the brain and spinal cord. • Myelitis: Inflammation of the spinal cord. • Meningitis: Inflammation of the membranes (meninges) that surround the brain and spinal cord. • Meningoencephalitis: Inflammation of the brain and meninges.
• Specimen • By lumbar puncture
• Microscopic Examination : • Smears are made from the sediment of centrifuged CSF. • Gram stain : intracellular gram-negative diplococci (meningococci) small gram-negative rods (H influenzae or enteric gram-negative rods). lancet-shaped gram-positive diplococci (pneumo-cocci) • Follow-Up Examination of Cerebrospinal Fluid: • glucose level and cell count toward normal is good evidence of adequate therapy. • Culture : • Sheep blood and chocolate agar together grow almost all bacteria and fungi that cause meningitis.
Urinary Tract • 1-Upper UTIs : kidneys and ureters. • 2-Lower UTIs : urinary bladder, the urethra, and in males, the prostate. • Cystitis: Inflammation of the urinary bladder. • Nephritis: inflammation of the kidneys. • Pyelonephritis: inflammation of the renal parenchyma. (the basic cellular tissue of the kidney).
• Proper Collection of urine Specimen: • 1. Have at hand a sterile, screw-cap specimen container and two to three gauze sponges soaked with nonbacteriostatic saline (antibacterial soaps for cleansing are not recommended). • 2. Spread the labia with two fingers and keep them spread during the cleansing and collection process. Wipe the urethra area once from front to back with each of the saline gauzes. • 3. Start the urine stream and, using the urine cup, collect a midstream specimen. Properly label the cup.
• Microscopic Examination • leukocytes, epithelial cells, and bacteria if more than 105/mL are present. • Culture • “urethral syndrome,” – ve culture with sign mean ureteral obstruction, tuberculosis of the bladder, gonococcal infection, or other disease must be considered.
Genital Tract • The urethra contain microflora in males and females, special female genital region supports the growth of many microorganisms. • Cervicitis: Inflammation of the cervix, that part of the uterus that opens into the vagina. • Endometritis: Inflammation of the endometrium (the inner layer of the uterine wall). • Oophoritis: Inflammation of an ovary.
Diseases A.Gonorrhea • A stained smear of a urethral or a cervical exudate that shows intracellular gram-negative diplococci strongly suggests gonorrhea. The sensitivity is about 90% for men and 50% for women Thus. • B. Syphilis • Dark-field or immunofluorescence examination of fresh tissue fluid expressed from the base of the chancre may reveal typical T. pallidum.
C. Vaginosis/Vaginitis • A-Bacterial vaginosis associated with Gardnerella vaginalis or Mobiluncus and parasite Trichomonas vaginalis. • The discharge : (1) is grayish and sometimes frothy, (2) has a pH above 4.6 (3) has an fishy odor (4) contains “clue cells,” large epithelial cells covered with gram-negative or gram-variable rods • B-Candida albicans vaginitis is diagnosed by finding yeast or pseudohyphae in a potassium hydroxide preparation of the vaginal discharge.
Oral Cavity • Dental caries: Tooth decay or cavities, Streptococcus mutans. • Gingivitis: Inflammation of the gingiva (gums). • Bacteria are isolated from oral swabs, Carefully collected scrapings or aspirates should be transported to the CML.
Gastrointestinal Tract • The gastrointestinal (GI) tract consists of a long tube with many expanded areas designed for digestion of food. • Most of the microorganisms low pH (1.5), and are inhibited from growing in the lower intestines by the resident microflora.
• Diarrhea: An abnormally frequent discharge of semi-solid or fluid fecal matter. • Dysentery: Frequent watery stools accompanied by abdominal pain, fever, and dehydration and may contain blood and/or mucus. • Enteritis: Inflammation of the intestines. • Gastritis: Inflammation of the mucosal lining of the stomach. • Gastroenteritis: Inflammation of the mucosal linings of the stomach and intestines.
Body Fluids • body fluid specimens include abdominal or peritoneal fluid, pleural or thoracentesis fluid, synovial (joint) fluid, and amniotic fluid. Carefully collected aspirates of amniotic and pleural fluids are usually transported to the CML in an anaerobic transport system.
Factors limiting usefulness of microbiology investigations Specimen should be obtained from site of infection Sample must be taken aseptically Sample size must be large enough Metabolic requirements for the organism must be maintained during sampling, storage, and transport. Wrong sample e.g. saliva instead of sputum Delay in transport / inappropriate storage e.g. CSF Overgrowth by contaminants e.g. blood cultures Insufficient sample / sampling error e.g. in mycobacterial disease Patient has received antibiotics
Laboratory Investigation of Microbial infections 1- Microscopy 2- Culture techniques 3- Biochemical reactions 4-GAS-LIQUID CHROMATOGRAPHY 5- Serological identification: 6- Molecular biology techniques 7- Bacteriophages 8-Animal pathogenicity
1-Microscopy • is a relatively simple and inexpensive, but much less sensitive method than culture for detection of small numbers of bacteria. • 1-simple microscope: is defined as a microscope containing only one magnifying lens.
• 2-compound microscope: is a microscope that contains more than one magnifying lens. • Because objects are observed against a bright background, or “bright field,” bright field microscope. • the regularly used condenser is replaced with what is known as a darkfield condenser, illuminated objects are seen against a dark background, or “dark field,” dark field microscope.
3-Electron Microscopes • Electron microscopes: use an electron beam as a source of illumination instead of visible light and magnets instead of lenses to focus the beam. • Transmission electron microscope: has a very tall column, at the top of which an electron gun fires a beam of electrons downward.
Microscopy Unstained preparations “Wet prep” Dark-ground illumination for syphilis Stained preparations Gram-stain Acid-fast stain Fluorescence Immunofluorescence
Gram stain
Principle laboratory diagnosis of infectious diseases
Acid-fast stain
Fluorescence
Immunofluorescence
Dark-ground illumination for syphilis
2-Culture • The media are referred to as artificial media or synthetic media because they do not occur naturally but are prepared in the laboratory. • Inoculation of a liquid medium involves adding a portion of the specimen to the medium. While Inoculation of a solid or plated medium involves the use of a sterile bacteriological loop to apply a portion of the specimen to the surface of the medium. • A CO2 incubator: has a cylinder of CO2 attached. CO2 is periodically introduced into the incubator to maintain a concentration of about 5% to 10%. • A non-CO2 incubator: contains room air; thus, it contains about 20% to 21% O2. • An anaerobic incubator: contains an atmosphere devoid of oxygen.
Culture • Solid media: are prepared by adding agar to liquid media and then pouring the liquid media into tubes or circular, shallow, plastic containers called petri dishes, where the media solidifies. • For Identification – Enumeration – sensitivity and chemical and products. Liquid media (broth):also known as broths, are usually contained in tubes and are thus often referred to as tubed media. • For enrichment or maximum sensitivity or measure amount of bacteria and motility.
Culture of Pathogenic Microbes • Most microbes of clinical importance can be grown, isolated, and identified with specialised growth media • 1-General Purpose Media • Support growth of most aerobic and facultatively aerobic organisms .EX: nutrient agar
• 2-enriched medium • medium containing a rich supply of special nutrients that promotes the growth of bacteria. EX: Blood agar • 3-Selective Media • contains substances that inhibit growth of certain organisms .EX: MacConkey agar • 4-Differential Media • permits the differentiation of organisms that grow on the medium. EX: MacConkey agar.
Antimicrobial Susceptibility Testing • 1-Disk Diffusion Test • Standard procedure for assessing antimicrobial activity • 2-E-TEST ( Epsilometer test) • strip contain more than one antibiotic • Inhibition Zones Used to determine an organism’s susceptibility to an antimicrobial agent
Minimum Inhibitory Concentration (MIC) • Antibiotic dilution to detect minimum concentration that prevent bacteria growth and disappear of turbidity.
• Catalase Test • This test is used to identify organisms that produce the enzyme catalase. H2O2 . EX: Staphylococcus spp • Oxidase Test • This test is used to identify microorganisms containing the enzyme cytochrome oxidase EX: Enterobacteriaceae 3-Biochemical reactions
• Coagulase test • Coagulase is an enzyme that clots blood plasma. EX: Staphylococcus aureus • bacitracin sensitivity testing • Distinguish between organisms sensitive to the antibiotic bacitracin and those not. EX: Streptococcus agalactiae (bacitracin resistant) and Streptococcus pyogenes (bacitracin sensitive).
• optochin sensitivity testing • This is a differential test used to distinguish between organisms sensitive to the antibiotic optochin and those not EX: Streptococcus pneumoniae (optochin sensitive) and other a- hemolytic streptococci (optochin resistant Streptococcus mitis) • MacConkey agar • This medium is both selective and differential. crystal violet which inhibit the growth of Gram-positive bacteria. The differential ingredient is lactose. Fermentation of this sugar results in an acidic pH and causes the pH indicator, neutral red, to turn a bright pinky-red color. Thus organisms capable of lactose fermentation such as Escherichia coli, form bright pinky-red colonies. MacConkey agar is commonly used to differentiate between the Enterobacteriaceae.
• Starch hydrolysis test • This test is used to identify bacteria that can hydrolyze starch using the enzymes a-amylase and oligo-1,6-glucosidase. EX: Bacillus subtilis • Methyl Red / Voges-Proskauer (MR/VP) • This test is used to determine which fermentation pathway is used to utilize glucose. EX: Escherichia coli is MR+ and VP-
CAMP Test • CAMP factor is a diffusible, heat-stable protein produced by group B streptococci. This is a synergistic test between Staphylococcus aureus and Streptococcus agalactiae. S. aureus produces sphingomyelin C, which binds to red blood cell membranes with CAMP.
• Urease test • This test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease. EX: Proteus mirabilis is a rapid hydrolyzer of urea • Motility agar • is a differential medium used to determine whether an organism is equipped with flagella and thus capable of swimming away from a stab mark
• Indole test
4-GAS-LIQUID CHROMATOGRAPHY • Two ways in which GLC can be used in the identification of bacteria and yeasts are : • (1) analysis of acid end products of metabolism. • (2) analysis of cellular fatty acids.
5-IMMUNODIAGNOSTIC PROCEDURES • Immunodiagnostic procedures (IDPs) are laboratory procedures that help to diagnose infectious diseases by detecting either antigens or antibodies in clinical specimens. • presence of antibodies to a particular pathogen: • 1. Present infection. • 2. Past infection: the person was infected with the pathogen in the past, and antibodies directed against that pathogen are still present in the person’s body • 3. Vaccination; the antibodies are the result of the person having been vaccinated against that particular pathogen at some time in the past
Principle laboratory diagnosis of infectious diseases
Serologicalmethods: • There are a variety of serological assays available including • Several of these tests can measure antibody titer by performing dilutions of patient serum to determine the lowest titer at which reactivity is seen. •1-ELISA.
2-agglutination
3-complement fixation (CF)
4-Direct and indirect immunofluorescence
Western Blot Immunoassays • This method is based on the electrophoretic separation of major proteins of the organism in question in a two-dimensional agarose gel.
6-Molecular Diagnostic Procedures • A. Nucleic Acid Hybridization Probes • the hybridization of a characterized nucleic acid probe to a specific nucleic acid sequence in a test specimen followed by detection of the paired hybrid. For example, single-stranded probe DNA (or RNA) is used to detect complementary RNA or denatured DNA in a test specimen.
•B-PCR •AmplificationsegmentofDNAorRNAmanytimestodetectit.
C. Bacterial Identification Using 16S rRNA Probe Hybridization • The 16S rRNA of each species of bacteria has stable (conserved) portions of the sequence. Many copies are present in each organism. Labeled probes specific for the 16S rRNA of a species are added, and the amount of label on the double stranded hybrid is measured. • EX: Histoplasma capsulatum
7-Bacteriophages • Bacteriophage (phage) are obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery. •1-generalizedtransduction •2-specializedtransduction
• 1-generalized transduction: A DNA fragment is transferred from one bacterium to another by a lytic bacteriophage that is now carrying donor bacterial DNA due to an error in maturation during the lytic life cycle.
• 2-specialized transduction: A DNA fragment is transferred from one bacterium to another by a temperate bacteriophage that is now carrying donor bacterial DNA due to an error in spontaneous induction during the lysogenic life cycle
8-Animal pathogenicity • use of non-human animals in experiments that seek to control the variables that affect the behavior or biological system under study. • Animals commonly used : guinea pigs, rabbits, mice • Importance of pathogenicity test: - • 1-Differentiate pathogenic and non pathogenic • 2- Isolation organism in pure form • 3- To test ability of toxin production • 4- Evaluation of vaccines and antibiotics
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Principle laboratory diagnosis of infectious diseases

  • 2. Outline Rejection of specimens 04 CLINICAL SPECIMENS 05 Laboratory Investigation of Microbial infections 06 COMMUNICATION BETWEEN PHYSICIAN AND LABORATORY 01 specimens 02 QUALITY ASSURANCE AND QUALITY CONTROL 03
  • 3. COMMUNICATION BETWEEN PHYSICIAN AND LABORATORY • The techniques used to characterize infectious depending on the clinical syndrome and the type of infectious agent. • Because no single test will permit isolation or characterization of all potential pathogens, clinical information is much more important for diagnostic microbiology than it is for clinical chemistry or hematology.
  • 4. Diagnosis of infectious diseases • The proper diagnosis of an infectious disease requires : • (1) patient history. • (2) physical examination of the patient. • (3) carefully evaluating the patient’s signs and symptoms. • (4) conducting appropriate laboratory tests and other methods. • (5) proper selection, collection, and transport of appropriate clinical specimens.
  • 5. Guidelines for the collection and transportation of specimens must emphasize two important aspects • I-Collection of the specimen before the administration of antimicrobial agents. • II-Prevention of contamination of the specimen with externally present organisms or normal flora of the body.
  • 6. The Three Components of Specimen Quality Component Explanation Proper selection of the specimen The specimen must be the appropriate type to diagnose the infectious disease suspected by the clinician. Proper collection of the specimen Proper collection eliminates or minimizes contamination of the specimen with microflora. Sometimes special collection devices are required. Proper transport of the specimen The specimen Transport let viable and/or preserves agent morphology (e.g., rapid transport, sometimes by packing the container in ice or by using a preservative)
  • 7. PROPER SELECTION, COLLECTION AND TRANSPORT OF CLINICAL SPECIMENS 1-The specimen must be properly selected. Collect the appropriate type of specimen for diagnosis of the infectious disease. 2-The specimen must be properly and carefully collected. 1-From a site of pathogen is most likely to be found. 2-Before antimicrobial therapy has begun 3-The acute stage is the best 4-Avoid harming the patient and causing discomfort. 5-Sufficient quantity. 6-All specimens should be placed or collected into a sterile container 3-Specimens must be properly transported to the lab Specimens should be protected from heat and cold to ensure represent the number and types of organisms present at the time of collection.
  • 8. QUALITY ASSURANCE AND QUALITY CONTROL IN THE CLINICAL MICROBIOLOGY LABORATORY •A-Quality Assurance (QA) •B-Quality Control (QC)
  • 9. Quality Assurance (QA) • It is continuously identify, monitor, evaluate, and improve the reliability
  • 10. Quality Control (QC) • Is designed to monitor the accuracy, reliability, and reproducibility of all tests performed within the laboratory and to identify and correct any problems that exist. • Components of a CML’s QC program include the following: • 1-Standard operating 2-Test verification. procedures manual. 3-Test methods and procedures. The procedures written for every aspect of CML work. use methods for most accurate The QC program must monitor, document, and evaluate all aspects of every test procedure that is performed in the CML.
  • 11. • 4-Media, reagents, staining solutions, antisera. • 5-Equipment and instruments. • 6-Records and reports. 7-Proficiency testing. Avoid expiration dates maintained and monitored of equipment for performance. QC measures and documented positive and negative controls used for every test procedure
  • 12. Criteria for rejection of specimens • i-Missing or inadequate identification. • ii-Insufficient quantity. • iii-collected in an inappropriate container. • iv-Contamination suspected. • v-Inappropriate transport or storage. • vi-Unknown time delay. • vii-Haemolysed blood sample.
  • 13. CLINICAL SPECIMENS FROM VARIOUS ANATOMICAL SITES AND ORGAN SYSTEMS • Circulatory System • Skin, Abscess, and Wound Specimens • Eyes and Ears • Respiratory System • Central Nervous System • Urinary Tract • Genital Tract • Oral Cavity • Gastrointestinal Tract • Body Fluids
  • 14. Circulatory System •Blood • Blood is usually sterile. • Bacteremia:presence of bacteria in the bloodstream. • Septicemia condition: bacteria product or any toxic substance in blood. • The severe types of septicemia are caused by Gram-negative bacilli that release endotoxin from their cell walls, it can induce fever and septic shock, which can be fatal.
  • 18. Lymphatic System • The lymphatic system consists of lymphatic vessels, lymphoid tissue (including lymph nodes, tonsils, thymus, and spleen), and lymph (the liquid that circulates through the lymphatic system),contains many lymphocytes. • specimens :culturing lymphnode and/or by blood culture, (if the pathogens have entered the bloodstream). • Lymphadenopathy: Diseased lymph nodes.
  • 19. Skin, Abscess, and Wound Specimens • skin is a type of host defense mechanism, serving as a physical barrier. • Sebaceous glands: Glands in the dermis that usually open into hair follicles and secrete an oily substance known as sebum. Folliculitis: Inflammation of a hair follicle Sty (or stye): Inflammation of a sebaceous gland that opens into a follicle of an eyelash. Furuncle: A localized pyogenic (pus-producing) infection of the skin, usually resulting from folliculitis; also known as a boil.
  • 20. Obtaining specimens from the skin: • 1-For pustules : • The covering removed using a sterile needle, then fluid and basal cells collected. • 2-For petechiae : • specimen is collected by vigorously scraping the outer margin of the lesion. • 3-Collecting abscess specimens: • the abscess contents are aspirated using a needle and syringe. • 4-Collecting wound specimens: • specimen should be an aspirate, or a sample taken from the advancing margin of the lesion.
  • 21. Eyes and Ears •Eyes • infectious diseases of the eye include the following: • Conjunctivitis: An infection or inflammation of the conjunctiva. • Keratitis: An infection or inflammation of the cornea.
  • 22. • Keratoconjunctivitis: An infection that involves both the cornea and conjunctiva. • specimen as conjunctival swab, corneal scraping, or aspirate. • Using separate sterile swabs, sample the conjunctiva of both eyes, even if only one eye is infected.
  • 23. •Ears • There are three pathways for pathogens to enter the ear: • (1) Eustachian tube, from the throat and nasopharynx. • (2) from the external ear. • (3) via blood or lymph. • otitis media Infection of the middle ear • otitis externa infection of the outer ear canal. • specimen collected by swab of external ear canal, fluid aspirated from the middle ear,
  • 24. Respiratory System Specimen: • A. Nasal swabs • B. Nasopharyngeal / Pernasal swabs • C. Nasopharyngeal aspirates • D. Nasal washings • E. Throat swabs • F. Lung aspirations • G. Open lung biopsy
  • 25. Central Nervous System • The nervous system is composed of the central nervous system (CNS) and the peripheral nervous system. • It is sterilized but agent enter through trauma, blood and lymph or along the peripheral nerves. • Glucose level indicator for infection. • Encephalitis: Inflammation of the brain. • Encephalomyelitis: Inflammation of the brain and spinal cord. • Myelitis: Inflammation of the spinal cord. • Meningitis: Inflammation of the membranes (meninges) that surround the brain and spinal cord. • Meningoencephalitis: Inflammation of the brain and meninges.
  • 26. • Specimen • By lumbar puncture
  • 27. • Microscopic Examination : • Smears are made from the sediment of centrifuged CSF. • Gram stain : intracellular gram-negative diplococci (meningococci) small gram-negative rods (H influenzae or enteric gram-negative rods). lancet-shaped gram-positive diplococci (pneumo-cocci) • Follow-Up Examination of Cerebrospinal Fluid: • glucose level and cell count toward normal is good evidence of adequate therapy. • Culture : • Sheep blood and chocolate agar together grow almost all bacteria and fungi that cause meningitis.
  • 28. Urinary Tract • 1-Upper UTIs : kidneys and ureters. • 2-Lower UTIs : urinary bladder, the urethra, and in males, the prostate. • Cystitis: Inflammation of the urinary bladder. • Nephritis: inflammation of the kidneys. • Pyelonephritis: inflammation of the renal parenchyma. (the basic cellular tissue of the kidney).
  • 29. • Proper Collection of urine Specimen: • 1. Have at hand a sterile, screw-cap specimen container and two to three gauze sponges soaked with nonbacteriostatic saline (antibacterial soaps for cleansing are not recommended). • 2. Spread the labia with two fingers and keep them spread during the cleansing and collection process. Wipe the urethra area once from front to back with each of the saline gauzes. • 3. Start the urine stream and, using the urine cup, collect a midstream specimen. Properly label the cup.
  • 30. • Microscopic Examination • leukocytes, epithelial cells, and bacteria if more than 105/mL are present. • Culture • “urethral syndrome,” – ve culture with sign mean ureteral obstruction, tuberculosis of the bladder, gonococcal infection, or other disease must be considered.
  • 31. Genital Tract • The urethra contain microflora in males and females, special female genital region supports the growth of many microorganisms. • Cervicitis: Inflammation of the cervix, that part of the uterus that opens into the vagina. • Endometritis: Inflammation of the endometrium (the inner layer of the uterine wall). • Oophoritis: Inflammation of an ovary.
  • 32. Diseases A.Gonorrhea • A stained smear of a urethral or a cervical exudate that shows intracellular gram-negative diplococci strongly suggests gonorrhea. The sensitivity is about 90% for men and 50% for women Thus. • B. Syphilis • Dark-field or immunofluorescence examination of fresh tissue fluid expressed from the base of the chancre may reveal typical T. pallidum.
  • 33. C. Vaginosis/Vaginitis • A-Bacterial vaginosis associated with Gardnerella vaginalis or Mobiluncus and parasite Trichomonas vaginalis. • The discharge : (1) is grayish and sometimes frothy, (2) has a pH above 4.6 (3) has an fishy odor (4) contains “clue cells,” large epithelial cells covered with gram-negative or gram-variable rods • B-Candida albicans vaginitis is diagnosed by finding yeast or pseudohyphae in a potassium hydroxide preparation of the vaginal discharge.
  • 34. Oral Cavity • Dental caries: Tooth decay or cavities, Streptococcus mutans. • Gingivitis: Inflammation of the gingiva (gums). • Bacteria are isolated from oral swabs, Carefully collected scrapings or aspirates should be transported to the CML.
  • 35. Gastrointestinal Tract • The gastrointestinal (GI) tract consists of a long tube with many expanded areas designed for digestion of food. • Most of the microorganisms low pH (1.5), and are inhibited from growing in the lower intestines by the resident microflora.
  • 36. • Diarrhea: An abnormally frequent discharge of semi-solid or fluid fecal matter. • Dysentery: Frequent watery stools accompanied by abdominal pain, fever, and dehydration and may contain blood and/or mucus. • Enteritis: Inflammation of the intestines. • Gastritis: Inflammation of the mucosal lining of the stomach. • Gastroenteritis: Inflammation of the mucosal linings of the stomach and intestines.
  • 37. Body Fluids • body fluid specimens include abdominal or peritoneal fluid, pleural or thoracentesis fluid, synovial (joint) fluid, and amniotic fluid. Carefully collected aspirates of amniotic and pleural fluids are usually transported to the CML in an anaerobic transport system.
  • 38. Factors limiting usefulness of microbiology investigations Specimen should be obtained from site of infection Sample must be taken aseptically Sample size must be large enough Metabolic requirements for the organism must be maintained during sampling, storage, and transport. Wrong sample e.g. saliva instead of sputum Delay in transport / inappropriate storage e.g. CSF Overgrowth by contaminants e.g. blood cultures Insufficient sample / sampling error e.g. in mycobacterial disease Patient has received antibiotics
  • 39. Laboratory Investigation of Microbial infections 1- Microscopy 2- Culture techniques 3- Biochemical reactions 4-GAS-LIQUID CHROMATOGRAPHY 5- Serological identification: 6- Molecular biology techniques 7- Bacteriophages 8-Animal pathogenicity
  • 40. 1-Microscopy • is a relatively simple and inexpensive, but much less sensitive method than culture for detection of small numbers of bacteria. • 1-simple microscope: is defined as a microscope containing only one magnifying lens.
  • 41. • 2-compound microscope: is a microscope that contains more than one magnifying lens. • Because objects are observed against a bright background, or “bright field,” bright field microscope. • the regularly used condenser is replaced with what is known as a darkfield condenser, illuminated objects are seen against a dark background, or “dark field,” dark field microscope.
  • 42. 3-Electron Microscopes • Electron microscopes: use an electron beam as a source of illumination instead of visible light and magnets instead of lenses to focus the beam. • Transmission electron microscope: has a very tall column, at the top of which an electron gun fires a beam of electrons downward.
  • 43. Microscopy Unstained preparations “Wet prep” Dark-ground illumination for syphilis Stained preparations Gram-stain Acid-fast stain Fluorescence Immunofluorescence
  • 50. 2-Culture • The media are referred to as artificial media or synthetic media because they do not occur naturally but are prepared in the laboratory. • Inoculation of a liquid medium involves adding a portion of the specimen to the medium. While Inoculation of a solid or plated medium involves the use of a sterile bacteriological loop to apply a portion of the specimen to the surface of the medium. • A CO2 incubator: has a cylinder of CO2 attached. CO2 is periodically introduced into the incubator to maintain a concentration of about 5% to 10%. • A non-CO2 incubator: contains room air; thus, it contains about 20% to 21% O2. • An anaerobic incubator: contains an atmosphere devoid of oxygen.
  • 51. Culture • Solid media: are prepared by adding agar to liquid media and then pouring the liquid media into tubes or circular, shallow, plastic containers called petri dishes, where the media solidifies. • For Identification – Enumeration – sensitivity and chemical and products. Liquid media (broth):also known as broths, are usually contained in tubes and are thus often referred to as tubed media. • For enrichment or maximum sensitivity or measure amount of bacteria and motility.
  • 52. Culture of Pathogenic Microbes • Most microbes of clinical importance can be grown, isolated, and identified with specialised growth media • 1-General Purpose Media • Support growth of most aerobic and facultatively aerobic organisms .EX: nutrient agar
  • 53. • 2-enriched medium • medium containing a rich supply of special nutrients that promotes the growth of bacteria. EX: Blood agar • 3-Selective Media • contains substances that inhibit growth of certain organisms .EX: MacConkey agar • 4-Differential Media • permits the differentiation of organisms that grow on the medium. EX: MacConkey agar.
  • 54. Antimicrobial Susceptibility Testing • 1-Disk Diffusion Test • Standard procedure for assessing antimicrobial activity • 2-E-TEST ( Epsilometer test) • strip contain more than one antibiotic • Inhibition Zones Used to determine an organism’s susceptibility to an antimicrobial agent
  • 55. Minimum Inhibitory Concentration (MIC) • Antibiotic dilution to detect minimum concentration that prevent bacteria growth and disappear of turbidity.
  • 56. • Catalase Test • This test is used to identify organisms that produce the enzyme catalase. H2O2 . EX: Staphylococcus spp • Oxidase Test • This test is used to identify microorganisms containing the enzyme cytochrome oxidase EX: Enterobacteriaceae 3-Biochemical reactions
  • 57. • Coagulase test • Coagulase is an enzyme that clots blood plasma. EX: Staphylococcus aureus • bacitracin sensitivity testing • Distinguish between organisms sensitive to the antibiotic bacitracin and those not. EX: Streptococcus agalactiae (bacitracin resistant) and Streptococcus pyogenes (bacitracin sensitive).
  • 58. • optochin sensitivity testing • This is a differential test used to distinguish between organisms sensitive to the antibiotic optochin and those not EX: Streptococcus pneumoniae (optochin sensitive) and other a- hemolytic streptococci (optochin resistant Streptococcus mitis) • MacConkey agar • This medium is both selective and differential. crystal violet which inhibit the growth of Gram-positive bacteria. The differential ingredient is lactose. Fermentation of this sugar results in an acidic pH and causes the pH indicator, neutral red, to turn a bright pinky-red color. Thus organisms capable of lactose fermentation such as Escherichia coli, form bright pinky-red colonies. MacConkey agar is commonly used to differentiate between the Enterobacteriaceae.
  • 59. • Starch hydrolysis test • This test is used to identify bacteria that can hydrolyze starch using the enzymes a-amylase and oligo-1,6-glucosidase. EX: Bacillus subtilis • Methyl Red / Voges-Proskauer (MR/VP) • This test is used to determine which fermentation pathway is used to utilize glucose. EX: Escherichia coli is MR+ and VP-
  • 60. CAMP Test • CAMP factor is a diffusible, heat-stable protein produced by group B streptococci. This is a synergistic test between Staphylococcus aureus and Streptococcus agalactiae. S. aureus produces sphingomyelin C, which binds to red blood cell membranes with CAMP.
  • 61. • Urease test • This test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease. EX: Proteus mirabilis is a rapid hydrolyzer of urea • Motility agar • is a differential medium used to determine whether an organism is equipped with flagella and thus capable of swimming away from a stab mark
  • 63. 4-GAS-LIQUID CHROMATOGRAPHY • Two ways in which GLC can be used in the identification of bacteria and yeasts are : • (1) analysis of acid end products of metabolism. • (2) analysis of cellular fatty acids.
  • 64. 5-IMMUNODIAGNOSTIC PROCEDURES • Immunodiagnostic procedures (IDPs) are laboratory procedures that help to diagnose infectious diseases by detecting either antigens or antibodies in clinical specimens. • presence of antibodies to a particular pathogen: • 1. Present infection. • 2. Past infection: the person was infected with the pathogen in the past, and antibodies directed against that pathogen are still present in the person’s body • 3. Vaccination; the antibodies are the result of the person having been vaccinated against that particular pathogen at some time in the past
  • 66. Serologicalmethods: • There are a variety of serological assays available including • Several of these tests can measure antibody titer by performing dilutions of patient serum to determine the lowest titer at which reactivity is seen. •1-ELISA.
  • 69. 4-Direct and indirect immunofluorescence
  • 70. Western Blot Immunoassays • This method is based on the electrophoretic separation of major proteins of the organism in question in a two-dimensional agarose gel.
  • 71. 6-Molecular Diagnostic Procedures • A. Nucleic Acid Hybridization Probes • the hybridization of a characterized nucleic acid probe to a specific nucleic acid sequence in a test specimen followed by detection of the paired hybrid. For example, single-stranded probe DNA (or RNA) is used to detect complementary RNA or denatured DNA in a test specimen.
  • 73. C. Bacterial Identification Using 16S rRNA Probe Hybridization • The 16S rRNA of each species of bacteria has stable (conserved) portions of the sequence. Many copies are present in each organism. Labeled probes specific for the 16S rRNA of a species are added, and the amount of label on the double stranded hybrid is measured. • EX: Histoplasma capsulatum
  • 74. 7-Bacteriophages • Bacteriophage (phage) are obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery. •1-generalizedtransduction •2-specializedtransduction
  • 75. • 1-generalized transduction: A DNA fragment is transferred from one bacterium to another by a lytic bacteriophage that is now carrying donor bacterial DNA due to an error in maturation during the lytic life cycle.
  • 76. • 2-specialized transduction: A DNA fragment is transferred from one bacterium to another by a temperate bacteriophage that is now carrying donor bacterial DNA due to an error in spontaneous induction during the lysogenic life cycle
  • 77. 8-Animal pathogenicity • use of non-human animals in experiments that seek to control the variables that affect the behavior or biological system under study. • Animals commonly used : guinea pigs, rabbits, mice • Importance of pathogenicity test: - • 1-Differentiate pathogenic and non pathogenic • 2- Isolation organism in pure form • 3- To test ability of toxin production • 4- Evaluation of vaccines and antibiotics
  • 78. Thank you Principle Laboratory Diagnosis of Infectious Diseases