This document discusses laboratory diagnosis of infectious diseases. It covers various topics including definitions of infection and immunity, manifestations of infectious processes, pathogenicity of pathogens and host immune reactions. It also discusses epidemiological processes, features of infectious diseases, common symptoms and signs, and various diagnostic techniques. These techniques include microscopy, culture techniques, biochemical reactions, serological identification, molecular biology techniques, bacteriophage typing, and specimen collection and processing.

1. LABORATORY DIAGNOSIS OF
INFECTIOUS DISEASES
Dr.Ihsan Edan Alsaimary
Department Of Microbiology,
College Of Medicine-university Of Basrah

2. Synopsis
 Infectious disease
( lemology,communicable disease) is:
① A clinical medicine
② A part of internal medicine
③ To study the regularity of the occurrence and development
of infectious disease in human body
④ To study etiology, pathogenesis, pathology, clinical
manifestation and the methods of diagnosis, treatment
and prevention for infectious disease.
⑤ In order to control spread of infectious disease in
population
⑥ Infectious disease is related to biology, immunology,
parasitological, epidemiology, pediatrics

3. Synopsis
 Infectious disease is:
① A group of common disease
② Caused by different pathogens
③ To possessing infectivity
④ To form epidemic
⑤ Infectious disease is a threat to the health of
people
⑥ The pathogens causing infectious diseases are:
virus, Chlamydia, rickettsia, mycoplasma,
spirochete, bacteria, fungus, protozoa and
helminthes

4. Infection and Immunity
 Definition of infection
① Complex process of interaction between
pathogen and human body
② Infection is composed of three factors:
pathogen, host and environment
③ There are commensalisms and
opportunistic infection

5. Infection and Immunity
Manifestations of infectious process (Infection
spectrum)
① Clearance of pathogen
② Covert infection (subclinical infection)
③ Overt infection (Clinical infection or apparent
infection)
④ Carrier state
Health carrier after covert infection
Convalescent carrier after overt infection
Incubatory carrier before onset of diseases
According to carrier time :
acute (transient) carrier
chronic carrier
⑤ Latent infection

6. Infection and Immunity
 The action of pathogen in
infectious process
The pathogenicity of pathogen is related
to :
Invasiveness
virulent
Number of pathogen
Mutation (variability)

7. Infection and Immunity
 The action of immune reaction of host in infectious
process
Non specific immunity
Barrier action (natural barrier)
External barrier:
skin , mucosa
Secretion of skin and mucosa
Accessory organ
Internal barrier: placenta, blood-brain
barrier
Phagocytosis
Humoral action :
Complement, Lysozyme, Fibronection,
Cytokines.

8. Infection and Immunity
 Specific immunity
Humoral immunity
Immunoglobulin: IgG, IgM, IgE,
IgA, IgD
Cell mediated immunity

9. Epidemic process and epidemic
factors of infectious disease
 Source of infection (basic conditions)
Patients (acute , chronic) ,Covert infection ,
Carrier ,Infected animal
 Route of transmission
Contact transmission ( direct and indirect) ,
Air-borne , Food water fly borne, Insects borne,
Blood borne ,Soil borne
 Susceptibility of population
 Factors of influencing epidemic process
nature factors ,social factors

10. Features of infectious disease
 Basic features
Pathogen
9 kinds of pathogen
Infectivity
Epidemiological features
Quality : exotic , local , endemicity
Quantity : sporadic occurrence, epidemic,
pandemic,
outbreak, endemicity, seasonal
Post infection immunity
Viral infection: life-long immunity
Bacteria infection: shorter immunity
Helminthes infection: no protective immunity
Protozoa infection: shorter immunity

11. Features of infectious disease
 Clinical features
Regularity in the development of
cource
Incubation period : diagnosis ,
qurantine period
Prodromal period
Period of apparent manifestation
Convalescent period
Relapse
Recrudescence

12. Common symptoms and signs
 Fever:
Three stages : effervescence
fastigium
deffervescence
Five kinds of fever: sustained fever,
remittent fever,
intermittent fever,
relapsing fever,
saddle type fever.
And irregular fever

13. Common symptoms and signs
 Rash eruption
Date of eruption
1st: chickenpox 2nd: scarlet fever
3rd: smallpox 4th: measles
5th: typhus 6th:typhoid fever
Location of eruption
Form of rash
Exanthema :maculo-papular rash
Petechia
Vesiculo-pustular rash
Ureicaria
Enanthema

14. Common symptoms and signs
 Toxemic symptoms
 Mononuclear phagocyte system
reactions
Hepato-splenomegale
Lymphonodus enlarged
 Clinical types
acute, subacute, mild, common,
severe,
fulminant, typical, atypical, abortive,
ambulatory

15. Diagnosis of infectious diseases
 *Epidemiological dates
 *Clinical features
Symptoms and signs
 *Laboratory findings
Routine examination of blood, urine, feces
Bio-chemical examinations
Etiological examinations
Direct exam
Isolation of pathogen
*Molecular biological examinations
* Immunological examinations
* Endoscope examinations
* Image examinations

16. Treatment of infectious disease
 General and supporting therapy
Isolation of patients, rest, diet,
nursing
 Pathogen or specific therapy
 Symptomatic therapy
 Rehabilitation
Physiotherapy acupuncture
 Chinese herbs or tradition medicine

17. Prevention of infectious disease
 Management of source of infection
35 kinds of notifiable infectious disease divided into
3 class
First class: 2 kinds. Reported 6h in city, 12h in country.
Second class: 24 kinds. reported 12h in city and country
Third class: 9 kind
 Cut off of route
Personal hygiene, public hygiene, insecticide,
disinfection
 Protect susceptible population
Actibe immunization
Passive immunization

18. Laboratory Investigation of Microbial infections
Examining specimens to detect isolate and identify pathogens:
1- Microscopy
2- Culture techniques
3- Biochemical reactions
4- Serological identification:
5- Molecular biology techniques
6- Bacteriophage typing

19. Specimen Selection, Collection, and
Processing
 The quantity material must be adequate
 Specimens are selected on the basis of signs and
symptoms, should be representative of the disease
process
 Contamination of the specimen must be avoided by
using only sterile equipment and aseptic
precautions
 The specimen must be taken to the laboratory and
examined promptly. Special transport media may
be helpful.
 Meaningful specimens to diagnose bacterial
infections must be secured before antimicrobial
drugs are administered.

20. 1- Microscopy
Microorganisms can be examined microscopically for:
a- Bacterial motility:
Hanging drop method:
A drop of bacterial suspension is placed between a cover slip and
glass slid
b- Morphology and staining reactions of bacteria:
Simple stain: methylene blue stain
Gram stain: differentiation between Gm+ve and Gm–ve bacteria
. Primary stain (Crystal violet)
. Mordant (Grams Iodine mixture)
. Decolorization (ethyl alcohol)
. Secondary stain ( Saffranin)
Ziehl-Neelsen stain: staining acid fast bacilli
. Apply strong carbol fuchsin with heat
. Decolorization (H2SO4 20% and ethyl alcohol
. Counter stain (methylen blue)

21. 2- Culture Techniques
* Culture media are used for:
- Isolation and identification of pathogenic organisms
- Antimicrobial sensitivity tests
* Types of culture media:
a- Liquid media:
- Nutrient broth: meat extract and peptone
- Peptone water for preparation sugar media
- Growth of bacteria detected by turbidity
b- Solid media:
- Colonial appearance
- Hemolytic activity
- Pigment production

22. Types of solid media
1- simple media:
Nutrient agar
2- Enriched media: media of high nutritive value
. Blood agar
. Chocolate agar
. Loffler’s serum
3- Selective media: allow needed bacteria to grow
. Lowenstein–Jensen medium
. MacConkeys agar
. Mannitol Salt Agar
4- Indicator media: to different. between lact. and non lact. ferment
. MacConkeys medium
. Eosine Methlyne blue Agar
5- Anaerobic media: for anaerobic cultivation
. Deep agar, Robertson’s Cooked Meat Medium

23. Colonial appearance on culture media
* Colony morphology:
. Shape . Size . Edge of colony . Color
* Growth pattern in broth:
. Uniform turbidity
. Sediment or surface pellicle
* Pigment production:
. Endopigment production (Staph. aureus)
. Exopigment production (Ps. aeruginosa)
* Haemolysis on blood agar:
. Complete haemolysis (Strept. Pyogenes)
. Partial haemolysis (Strept. Viridans)
* Growth on MacConkey’s medium:
. Rose pink colonies (Lactose fermenters)
. Pale yellow colonies (Non lactose fermenters)

24. 3- Biochemical Reaction
Use of substrates and sugars to identify pathogens:
a- Sugar fermentation:
Organisms ferment sugar with production of acid only
Organisms ferment sugar with production of acid and gas
Organisms do not ferment sugar
b- Production of indole:
Depends on production of indole from amino acid tryptophan
Indole is detected by addition of Kovac’s reagent
Appearance of red ring on the surface
e- H2S production:
Depends on production H2S from protein or polypeptides
Detection by using a strip of filter paper containing lead acetate

25. 3- Biochemical Reaction (cont.)
c- Methyl red reaction (MR):
Fermentation of glucose with production of huge amount of acid
Lowering pH is detected by methyl red indicator
d- Voges proskaur’s reaction (VP):
Production of acetyl methyl carbinol from glucose fermentation
Acetyl methyl carbinol is detected by addition KOH
Color of medium turns pink (positive)
e- Action on milk:
Fermentation of lactose with acid production
Red color if litmus indicator is added

26. 3- Biochemical Reaction (cont.)
f- Oxidase test:
Some bacteria produce Oxidase enzyme
Detection by adding few drops of colorless oxidase reagent
Colonies turn deep purple in color (positive)
g- Catalase test:
Some bacteria produce catalase enzyme
Addition of H2O2 lead to production of gas bubbles (O2 production)
h- Coagulase test:
Some bacteria produce coagulase enzyme
Coagulase enzyme converts fibrinogen to fibrin (plasma clot)
Detected by slide or test tube method
i- Urease test:
Some bacteria produce urease enzyme
Urease enzyme hydrolyze urea with production of NH3
Alklinity of media and change color of indicator from yellow to pink

27. 4- Animal pathogenicity
* Animal pathogenicity test:
Animals commonly used are guinea pigs, rabbits,
mice
* Importance of pathogenicity test:
- Differentiate pathogenic and non pathogenic
- Isolation organism in pure form
- To test ability of toxin production
- Evaluation of vaccines and antibiotics

28. Serological identification
A- Direct serological tests:
- Identification of unknown organism
- Detection of microbial antigens by using specific
known antibodies
- Serogrouping and serotyping of isolated organism
B- Indirect serological tests:
- Detection of specific and non specific antibodies
(IgM & IgG) by using antigens or organisms

29. DIAGNOSTIC TECHNOLOGIES
 Immunoserology
• Hemagglutination
• EIA
• Latex agglutination
• Complement fixation
• Immunoflorecent

30. RAPID DIAGNOSTIC TESTS
 High sensitivity and specificity
 High negative and positive predictive values
 High accuracy compared to gold standard
 Simple to perform
 Rapid turn around time
 Cost effective

31. LIMITATIONS OF CONVENTIONAL
CLINICAL MICROBIOLOGY
 Culture
• Labor intensive
• Need for special media
• Prolonged period of time to culture
• Some organisms are uncultivable on artificial media
• Potential health hazards
 Antigen Detection
• Negative tests require confirmation
• Effected by poor specimen collection
• Low microbe burden
 Serology
• Unhelpful during early stage of infection
• Not quite useful in immunocompromised patients

32. Molecular Biology Techniques
A- Genetic probes (DNA or RNA probes):
Detection of a segment of DNA sequence (gene) in unknown
organism using a labeled probe
Probe: consists of specific short sequence of labeled single-
stranded DNA or RNA that form strong covalently
bonded hybrid with specific complementary strand of
nucleic acid of organism in question
B- Polymerase chain reaction (PCR):
Amplification of a short sequence of target DNA or RNA Then
It is detected by a labeled probe
C- Plasmid profile analysis:
Isolation of plasmids from bacteria and determination of their
size and number compared with standard strains by agarose
gel electrophoresis

33. MOLECULAR DIAGNOSTICS
 Most widely used is PCR
• High sensitivity
• High specificity
• Diversity
 Nucleic acid probes
• Do not amplify DNA

34.  *Multiplex PCR
• Uses single clinical specimen to investigate several
potential pathogens simultaneously
 Encephalitis/meningitis panel: HSV,VZV, CMV HHV-6, EBV,
Enteroviruses
 *Real-time PCR
• Utilizes a fluorescent labeled probe
• Requires small volumes thus takes 30-60 minutes to
complete
Polymerase Chain Reaction
*Specific PCR: Uses primers to known DNA targets.
Use when conventional diagnostics are inadequate, time
consuming, difficult and hazardous
*Broad range PCR: uses complementary primers to
conserved regions shared by a given taxonomic group
Used in cases of B. henselae and Mycobacterium spp

35. Real time PCR for Diagnosis of
Infectious Disease
Detect PCR product during synthesis
Requires fluorescence-based detection and
specialized detection instrumentation
Advantages
Less time for results
Improved analytical sensitivity

36. OTHER USES OF MOLECULAR DIAGNOSTICS
 Viral load monitoring
 Viral genotyping
 Bacterial resistance detection
 Bacterial genotyping

37. LIMITATION OF PCR TECHNOLOGIES
 Specimen should be frozen until
amplification
 No antimicrobial sensitivity is available
 Needs the clinician to name the suspect
 Cost
 False positives caused by amplification of
contaminants
 Only sample from normally sterile sites
should be considered for broad-range
PCR
 Specimen is required to be refrigerated or
stored in alcohol before processing

38. Bacteriophage

39. Bacteriophage
Bacteriophages are viruses
that parasitize bacterial cell
Replication of Bacteriophage :
A- Lytic or vegetative cycle:
End by lysis of bacterial cell and release of copies of the phage
1) Adsorption:
Adsorption occurs between attachment sites on the phage (tail
fibres) and specific receptor sites on bacteria
It is specific strep (sensitivity of bacteria to different phages)
2) Penetration:
The tail sheath will contract and inject DNA into bacterial cell

40. A- Lytic or Vegetative Cycle
3) Eclipse phase:
Viral DNA directs the host cell metabolism to synthesize new
enzymes and proteins for phage synthesis
4) Intracellular synthesis:
Host cell machinery is directed by genetic information provided
by phage nucleic acid to synthesize phage coats and nucleic a.
5) Assembly:
Protein subunits of the phage head and tail aggregate
Each capsid acquires nucleic acid molecule to become a mature
phage particle
6) Release:
Accumulation of huge number of phage
The cell bursts and phage particles are released

41. II- Temperate Phage cycle “Lysogenic cycle”
* Adsorption and penetration take place as in lytic cycle
* Virus DNA integrate with host chromosome (Prophage)
and replicate as part of host chromosome
* The bacterial cell is called a”lysogenic bacterium”
* Lysogenic bacterium has certain characters:
a- Immune to infection by another phage
b- Acquire new properties e.g. production of exotoxin
Diphtheria bacilli, Cl. Botulinum, Strpt. Pyogen
erthrogenic toxin

42. Outcome of Temperate cycle
1) The cell continue carrying prophage indefinitely,
passing it to daughter cells
2) The prophage detach from the bacterial
chromosome and start a lytic cycle
3) As prohage is detached it may carry genetic
material of bacterial chromosome
As it infects another bacterium , it will transmit to
it new characters

43. Practical applications using phages
* Phages are important as a research tools
* Phages are used as vectors in DNA
recombinant technology
* Phage typing of bacteria is important in
tracing source of infection for
epidemiologic purposes

44. Antimicrobial Susceptibility testing
 Introduction:
 Identification of a bacterial isolate from a patient
provides guidance in the choice of an appropriate
antibiotic for treatment
 Many bacterial species are not uniformly
susceptible to a particular anti-bacterial
compound
 This is particularly evident among the
Enterobacteriaceae, Staphylococcus spp., and
Pseudomonas spp.
 The wide variation in susceptibility and high
frequencies of drug resistance among strains in
many bacterial species necessitates the
determination of levels of resistance or
susceptibility as a basis for the selection of the
proper antibiotic for chemotherapy

45.  Antimicrobial Susceptibility testing can be
down by three ways:
1.Minimum Inhibitory Concentration (MIC)
2.Disk Diffusion Method
3.Minimum Bactericidal Concentration
(MBC)

46.  Most important aspect of laboratory
medicine
• Insufficient quantity
• Contamination
• Improper transport media
• Delay in transportation
• Inappropriate storage