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

2. COLLECTION AND PROCESSING OF CLINICAL SPECIMEN
Most important aspect of laboratory medicine
Insufficient quantity
Contamination
Improper transport media
Delay in transportation
Inappropriate storage

3. COLLECTION AND PROCESSING OF CLINICAL SPECIMEN
Collecting Blood
Clean with 70% ethyl alcohol
Disinfect with 10% povidone-iodine
Allow to dry for at least 1 minute
No wiping!
Clean the rubber stopper of the bottle
Use alcohol for Bactec bottle to prevent cracking

4. COLLECTION AND PROCESSING OF CLINICAL SPECIMEN
Collect enough blood
1-2ml in neonate
2-3ml in infants
3-5ml in children
10-20ml in adolescent
Rapid inoculation
A 3 hour delay result in 25% reduction in recovery of S. pneumoniae
Paisley JW, Lauer BA. Pediatric blood cultures. Clin Lab Med 1994; 14: 17
Roback MG, Tsai AK, Hanson KL. Delayed incubation of blood culture bottles: Effect on recovery rate of S. pneumoniae. Pediatr Emerg Care 1994; 10: 268

5. COLLECTION AND PROCESSING OF CLINICAL SPECIMEN
Collecting urine
Clean-voided midstream urine
Use of urine bag
Catheterized specimen/ Suprapubic aspiration
Collecting CSF
CSF is hypotonic
Refrigeration can render fastidious bacteria non-viable
Cell count decreases by 32% after 1 hour and 50% after 2 hours
Steele RW, Mormer DJ, O’Brien MD, et al. Leukocyte survival in cerebrospinal fluid. J Clin Microbiol 1986; 23: 965

6. COLLECTION AND PROCESSING OF CLINICAL SPECIMEN
Insufficient quantity/quality
Small quantity for optimal analysis
Poor specimen e.g. eye cultures for chlamydiae should have enough cellular element
Contamination
During collection
During transport
Contamination in the lab

7. COLLECTION AND PROCESSING OF CLINICAL SPECIMEN
Improper transport media
Prevent drying
Maintain optimal physiochemical environment
Prevent oxidation and destruction of enzymes
Provide adequate nutrients
Three major culture media
Enrichment: chocolate and sheep blood
Selective: TCBS, mannitol salt medium
Differential: MacConkey-ability to ferment lactose

8. COLLECTION AND PROCESSING OF CLINICAL SPECIMEN
Delay in transportation
Holding conditions are specimen or pathogen specific
Urine: 2˚ C to 8˚C
Inoculated blood: 35˚ C to 37˚C

9. SPECIFIC EXAMPLES
Specimen for isolation of N. gonorrhoeae should be inoculated into a specific media, transported within 30 minutes of collection, incubated at 35˚-37˚C in 5-10% co2
Stool for ova & parasite should be placed in preservatives
CSF is held in room temperature and never refrigerated
Stool for C. difficle must be refrigerated or frozen

11. 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

12. 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 ( Safranin)
Ziehl-Neelsen stain: staining acid fast bacilli
. Apply strong carbol fuchsin with heat
. Decolorization (H2SO4 20% and ethyl alcohol
. Counter stain (methylen blue)

13. 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

14. Types of solid media Nutrient agar
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

15. 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)

16. 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

17. 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

18. 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

19. 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

20. 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

21. DIAGNOSTIC TECHNOLOGIES
Antigen Detection Assays
Most has poor sensitivity and specificity
57% sensitivity and 98% specificity for pneumococcal pneumonia
Conc. Urine EIA for Legionella pneumophila serogroup 1 has 89% sensitivity and 100% specificity
Immunochromatographic assay has better sensitivity and are faster

22. DIAGNOSTIC TECHNOLOGIES
Immunoserology
Hemagglutination
EIA
Latex agglutination
Complement fixation
Immunoflorecent

23. 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

24. Respiratory Syncytial Virus RSV Immunoassay
Pathogens
Generic name of test
Mechanism
Sensitivity
(%)
Specificity
Positive
Predictive
Value (%)
Negative
Time to perform test
Respiratory Syncytial Virus
RSV Immunoassay
Qualitative detection of RSV antigen by immunoassay
89-93
96-97
80-89
15 minutes
Influenza A Virus
Rapid Flu A
Detection of influenza A nucleoprotein antigen
78-82
80-91
90-97
10-15 minutes
Influenzas B Virus
Rapid Flu B
Detection of influenza B nucleoprotein antigen
58-71
Not available
Influenza A & B Viruses
Rapid Flu A & B
Non-differential detection of both influenza A & B by neuraminidase enzyme assay
52-73
92-99
95-98
74-80
22 minutes
Influenza A + B Viruses
Influenza A + B
Differential detection of both influenza A & B by Immunoassay
72 – 82
96 – 99
80-90
10 minutes
Epstein Barr Virus
Mono Spot
Detection of heterophile antibodies
91-99 96
97-98 99
3-5 minutes
HIV
Rapid HIV
Detects HIV-1 antibody
10-20 minutes

25. Pathogens
Mechanism
Sensitivity
(%)
Specificity
Positive
Predictive
Value (%)
Negative
Time to perform test
Group A Streptococcus
Detects group A staphylococcal carbohydrate antigen by immunoassay
89-94
(Compared to culture)
95-99
55-89
90-97
5 minutes
Helicobacter pylori
Detects immunoglobulin G antibodies specific to H. pylori
85-90
(Compared to biopsy)
80-89 85 79
5-10 minutes
Borrelia burgdorferi
Detects antibodies to B. burgdorferi using recombinant antigen 72
(Compared to ELISA) 97
Not available
20 minutes

26. 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

27. 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

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

29. MOLECULAR DIAGNOSTICS
Polymerase Chain Reaction
Specific PCR: Uses primers to known DNA targets. So far 31 clinical bacterial gene sequence are known and 38 in progress
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

30. MOLECULAR DIAGNOSTICS
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 minutes to complete

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

32. LIMITATION OF PCR TECHNOLOGIES
Specimen should be frozen until amplification
No antimicrobial sensitivity is available
Needs the clinician to name the suspect

33. LIMITATION OF PCR TECHNOLOGIES
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

34. 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

35. 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

36. Antimicrobial Susceptibility testing can be down by three ways:
Minimum Inhibitory Concentration (MIC)
Disk Diffusion Method
Minimum Bactericidal Concentration (MBC)

37. 1. Minimum Inhibitory Concentration (MIC) :
Principle:
The tube dilution test is the standard method for determining levels of resistance to an antibiotic.
Serial dilutions of the antibiotic are made in a liquid medium which is inoculated with a standardized number of organisms and incubated for a prescribed time.
The lowest concentration of antibiotic preventing appearance of turbidity is considered to be the minimal inhibitory concentration (MIC).

38. Different concentrations of Gentamycin in Nutrient broth:
Conc. in mcg/ml
Gentamicin, generally considered a bacteriocidal antibiotic, for this bacterium, has an MIC of 0.8 mcg/ml

39. Different concentrations of Tetracycline in Nutrient broth:
Conc. in mcg/ml
Tetracycline, generally considered a bacteriostatic antibiotic, for this bacterium, has an MIC of 1.6 mcg/ml

40. 2. Disk-diffusion Method (Kirby-Bauer Method):
The disk-diffusion method (Kirby-Bauer) is more suitable for routine testing in a clinical laboratory where a large number of isolates are tested for susceptibility to numerous antibiotics.
An agar plate is uniformly inoculated with the test organism
A paper disk impregnated with a fixed concentration of an antibiotic is placed on the agar surface.
Growth of the organism and diffusion of the antibiotic commence simultaneously resulting in a circular zone of inhibition in which the amount of antibiotic exceeds inhibitory concentrations.
The diameter of the inhibition zone is a function of the amount of drug in the disk and susceptibility of the microorganism.

41. This test must be rigorously standardized since zone size is also dependent on:
inoculum size,
medium composition,
temperature of incubation,
excess moisture and
thickness of the agar.
Zone diameter can be correlated with susceptibility as measured by the dilution method.
Further correlations using zone diameter allow the designation of an organism as "susceptible", "intermediate", or "resistant" to concentrations of an antibiotic which can be attained in the blood or other body fluids of patients requiring chemotherapy.

42. Using a dispenser, antibiotic-impregnated disks are placed onto the agar surface.
As the bacteria on the lawn grow, they are inhibited to varying degrees by the antibiotic diffusing from the disk.

43. Staphylococcus aureus (MRSA)
Note the yellowish pigmentation of the bacterial lawn, and the lack of inhibition by the Oxacillin disk

44. Streptococcus pneumoniae (Pneumococcus):
The brownish tint of the blood agar plate outside the zones of bacterial inhibition is caused by alpha-haemolysis.

45. Pseudomonas aeruginosa:
The greenish tint of the lawn and plate in general is caused by the diffusible pigment made by the Pseudomonas aeruginosa itself.

46. Examples : case study examination

47. Nasopharyngeal culture for herpes simplex
You are asked to attend the delivery of a term infant because the baby is small for gestational age, and prenatal ultrasonography revealed periventricular cerebral calcifications. The infant’s birth weight is 2,000 g. On physical examination, you note hepatosplenomegaly and a petechial rash on the face and trunk.
Of the following, the BEST laboratory test for diagnosing the cause of these findings is
Nasopharyngeal culture for herpes simplex
Rapid plasma reagin for syphilis
Serum immunoglobulin (Ig) G titer for rubella
Serum IgM titer for toxoplasmosis
Urine culture for cytomegalovirus
57 E. Culture allows for diagnosis of unsuspecting viruses

48. Direct fluorescent antibody for Bordetella pertussis
A 12-year-old girl comes to your office complaining of headache, malaise, fever to 101 F (38.3  C), rhinorrhea, and a sore throat for the past 3 to 4 days. Most recently, she developed hoarseness and cough. On physical examination, you note crackles and wheezes throughout the lung fields. Chest radiography demonstrates interstitial infiltrates in the lower lung fields bilaterally.
Of the following, the BEST test to order to confirm the patient’s diagnosis is:
Direct fluorescent antibody for Bordetella pertussis
Enzyme immunoassay for respiratory syncytial virus
Serology for Mycoplasma pneumoniae
Throat culture for group A Streptococcus
Viral culture for parainfluenza
63 C

49. Mantoux skin test with purified protein derivative Monospot
A 16-year-old boy presents with a 5-day history of low-grade fever, headache, mild nasal congestion, and a persistent cough associated with posttussive syncope, he reports that it is hard for him to catch his breath after one of his coughing episodes. His immunizations are up to date. Several of his classmates are ill with similar symptoms. Chest radiography results are normal.
Of the following, the test that is MOST likely to aid in the diagnosis of this patient is:
Cold agglutinin test
Mantoux skin test with purified protein derivative
Monospot
Pertussis direct fluorescent antibody
Sputum Gram stain
70 D

50. Of the following, the BEST diagnostic test for this child is:
A 5-year-old girl develops fever, swelling of the parotid gland, and headache.
Of the following, the BEST diagnostic test for this child is:
Bacterial culture of parotid duct secretions
Epstein-Barr virus serology
Mumps serology
Serum amylase
Viral culture of respiratory secretions C

51. Gastric aspirates for culture Needle aspiration of the node
A 3-year-old boy presents with a large, non-tender, rubbery anterior cervical lymph node. You prescribe a course of dicloxacillin, but there is no change in the node. Results of Mantoux purified protein derivative skin test reveal 8 mm in duration.
Of the following, the BEST diagnostic procedure to undertake in this patient now is:
Biopsy of the node
Chest radiography
Excision of the node
Gastric aspirates for culture
Needle aspiration of the node
C. Atypical mycobacterium

52. Cytology of a conjunctival swab Rapid plasma reagin test
A newborn has evidence of symmetric intrauterine growth restriction. Evaluation reveals microcephaly with intracranial calcifications, “salt and pepper” retinopathy, hearing deficit, enlarged liver and spleen, and purpura. Laboratory evaluation documents thrombocytopenia.
Of the following, the test MOST likely to confirm the diagnosis in this infant is:
Cytology of a conjunctival swab
Rapid plasma reagin test
Serology of blood
Urine assay for interferon
Viral culture of urine C

53. Calcofluor white stain Giemsa stain Gram stain Kinyoun stain
Of the following, the BEST direct stain to detect Mycobacterium tuberculosis is the
Calcofluor white stain
Giemsa stain
Gram stain
Kinyoun stain
Periodic acid-Schiff stain D

54. Of the following, the test that would BEST confirm the diagnosis is:
A 9-year-old boy has had a nonproductive cough for the past 3 weeks. He has been afebrile and otherwise feeling well. On physical examination, you note widespread rales. Chest radiography reveals bilateral, diffuse infiltrates. You diagnose pneumonia, most likely due to Mycoplasma pneumoniae.
Of the following, the test that would BEST confirm the diagnosis is:
Bacterial culture of sputum
Blood culture
Gram stain of sputum
Mycoplasma-specific immunoglobulin M
Serum cold agglutinins D

55. Bacterial culture of cerebrospinal fluid of herpes simplex
A 5-year-old boy is hospitalized in January with fever and seizures. Lumbar puncture reveals clear cerebrospinal fluid that has a white blood cell count of 47/ cu mm, all of which are lymphocytes. On physical examination, he appears obtunded but arouses with painful stimuli. Neurologic examination reveals no focal findings.
Of the following, the diagnostic test that is MOST likely to reveal the etiology of this child’s illness is:
Bacterial culture of cerebrospinal fluid of herpes simplex
Polymerase chain reaction test of cerebrospinal fluid for herpes simplex
Streptococcus pneumoniae bacterial antigen test of cerebrospinal fluid
Viral culture of cerebrospinal fluid
Viral culture of nasopharyngeal and rectal swabs

56. Maternal human immunodeficiency virus serology
A newborn has hepatosplenomegaly, purpuric rash, jaundice, thrombocytopenia, and microcephaly. Computed tomography of the head demonstrates cerebral calcifications
Of the following, the MOST appropriate diagnostic testing for this infant includes:
Maternal human immunodeficiency virus serology
Serologic testing of mother and infant for cytomegalovirus
Serologic testing of mother and infant for Toxoplasma
VDRL on infant and maternal sera
Viral culture of swabs of infant’s throat and conjunctivae

57. A 12-year-old girl is brought to your office of evaluation following 4 weeks of diarrhea, abdominal pain, and weight loss. You suspect giardiasis.
Of the following, the MOST reliable next step to establish the diagnosis is to examine a single stool sample for:
Giardia antigen
Leukocytes
Ova and parasites pH
Reducing substances

58. Of the following, the BEST test to establish the diagnosis is:
A 4-month-old infant develops severe paroxysmal coughing 10 days after the onset of nasal congestion and rhinorrhea. His mother reports that often 15 to 20 coughs occur in rapid succession.
Of the following, the BEST test to establish the diagnosis is:
Bronchoscopy that demonstrates the presence of a foreign body
Culture of a nasal swab that grows a small gram-negative coccobacillus
Culture of a nasal swab that shows viral growth
pH probe that demonstrates gastroesophageal reflux
Skin testing with demonstration of allergies B