Principle laboratory diagnosis of infectious diseases by different methods

1. Principle Laboratory
Diagnosis of
Infectious Diseases
M.S.C. Microbiology student Murtadha Ali Hadi

2. Outline
Rejection of specimens04
CLINICAL SPECIMENS05
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.

15. phlebotomy

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

44. Gram stain

46. Acid-fast stain

47. Fluorescence

48. Immunofluorescence

49. Dark-ground illumination for syphilis

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

62. • Indole test

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.

67. 2-agglutination

68. 3-complement fixation (CF)

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.

72. •B-PCR
•AmplificationsegmentofDNAorRNAmanytimestodetectit.

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.
• It’s used to detect of bacteria because there is types specialized for some bacteria to lysis
it.as examples Phage adsorption to the S. aureus cell is the first critical step in phage
replication.
• Types of bacteriophage:
•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 and rat.
• 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 youPrinciple Laboratory Diagnosis of Infectious Diseases