Laboratory diagnosis of infectious disease in microbiology

1. LABORATORY DIAGNOSIS OF
INFECTIOUS DISEASES
Dr. Zakir Hossain Habib
Principal Scientific Officer
Department of Microbiology
Institute of Epidemiology, Disease Control &Research (IEDCR)
Dhaka, Bangladesh

2. 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

3. Microbiology specimen selection and collection are the
responsibility of the medical staff, not usually the
laboratory, although…….
The certified specialist may be called upon for consultation
or assistance.

4. At an elementary level, the physician needs answers to 3
very basic questions from the laboratory:
Is my patient’s illness caused by a microbe?
If so, what is it?
What is the susceptibility profile of the organism so
therapy can be targeted?

5. Specimen
A properly collected specimen is the single most
important step.
Selection timing method of collection
Recovery of organism is most significant
if ……………….
agent is isolated from a normally sterile site.

6. General rules about specimen
Quantity must be adequate
Sample should be representative
Contamination of specimen must be avoided.
Specimen should be taken promptly and examined.
Specimens must be taken before antimicrobial drugs are
administered.

7. Which specimen?
According to presenting clinical picture
If symptom and signs point to one organ system-specimen
should be obtained from that source.
In the absence of localizing signs
-repeated blood samples for culture.

8. The laboratory requires a specimen, not a swab of a
specimen.
Actual tissue, aspirates, and fluids are always specimens of
choice, especially from surgery.
Swabs are expected from nasopharyngeal and viral
respiratory infections.

9. The importance of normal flora
M. tuberculosis
Salmonella typhi -----ALWAYS PATHOGEN
Brucella species.
Many infections are caused by members of normal flora.
The relative number of specific organisms found in a
culture are important in that case
e.g. Klebsiella pneumoniae in sputum culture

10. Laboratory procedures for diagnosis of infectious
diseases
1 Morphologic identification
2. Culture isolation
3. Detection of antigen by immunologic (latex agglutination,
enzyme immunoassay or EIA, etc) or fluorescein- labeled
antibody stain
4. DNA-DNA or DNA-RNA hybridization
5. Detection and amplification of organism nucleic acid
6. Demonstration of meaningful antibody or cell mediated
immune responses in an infectious agent

11. Wet mount:
The simplest method.
Example, to examine CSF for the presence of Cryptococcus
neoformans, with India ink as a background against which to
visualize large-capsuled yeast cells.
Dark-field illumination:
Spirochetes - from genital lesions
Borrelia or Leptospira - in blood.

12. Wet mount:
Skin scrapings and hair samples can be examined with the use
of either:
10% KOH wet-mount preparations
or
Calcofluor white method and ultraviolet illumination to
detect fungal elements as fluorescing structures.
Staining of wet mounts—e.g., with lactophenol cotton blue
stain for fungal elements—often is used for morphologic
identification.

13. Gram stain
Particularly useful for examining sputum for
polymorphonuclear leukocytes (PMNs) and bacteria.
Sputum specimens with 25 PMNs and <10 epithelial cells per
low-power field often provide clinically useful information.
However, the presence in "sputum" samples of >10 epithelial
cells per low-power field and of multiple bacterial types
suggests contamination with oral microflora.

14. Gram stain
The examination of CSF and joint, pleural, or peritoneal
fluid with Gram's stain -bacteria and/or PMNs are present.
>104 bacteria per milliliter should be detected.
Centrifugation often is performed before staining to
concentrate specimens
This simple method is particularly useful for examination of
CSF for bacteria and white blood cells or
examination of sputum for mycobacteria.

15. Despite the difficulty of discriminating between normal
microflora and pathogens, Gram's stain may prove useful for
specimens from areas with a large resident microflora if a
useful biologic marker (signal) is available.
Gram's staining of vaginal swab specimens can be used to
detect epithelial cells covered with gram-positive bacteria
in the absence of lactobacilli and the presence of gram-
negative rods—a scenario regarded as a sign of bacterial
vaginosis.
Similarly, examination of stained stool specimens for
leukocytes is useful as a screening procedure before testing
for Clostridium difficile toxin or other enteric pathogens.

16. Gram stain:
Presence of microflora may complicate Gram stain
diagnosis.
Gram's staining of vaginal swab-
- Epithelial cells covered with Gram-
positive bacteria
- Absence of lactobacilli
- Presence of gram-negative rods
Bacterial vaginosis

17. Ziehl-Neelsen stain:
The acid-fast stain identifies organisms that retain carbol
fuchsin dye after acid/organic solvent disruption (e.g.,
Mycobacterium spp).
Modifications of this procedure allow the differentiation of
Actinomyces from Nocardia or other weakly (or partially)
acid-fast organisms.
The acid-fast stain is applied to sputum, other fluids, and
tissue samples when acid-fast bacilli (AFB, e.g.,
Mycobacterium species) are suspected.

18. Fluorochrome Stains
Fluorochrome stains such as
-Acridine orange (nucleic acid)
-Auramine-rhodamine (mycobacteria)
-Calcofluor white (fungal cell wall)
Capsular, flagellar, and spore stains are used for identification
or demonstration of characteristic structures.

19. Immunofluorescent Stains
The direct immunofluorescent antibody technique uses
antibody coupled to a fluorescent compound (e.g.,
fluorescein) and directed at a specific antigenic target to
visualize organisms or subcellular structures.
The fluorescing compound absorbs ultraviolet light and
reemits light at a higher wavelength that is visible to the
human eye.

20. Principles of fluorochroming and immunofluorescence

21. Acid fast bacilli
Comparison of Z-N stained and auramine-rhodamine stained
Mycobacterium spp.

22. Indirect immunofluorescent antibody tech.
An unlabeled (target) antibody binds a specific antigen. The
specimen is then stained with fluorescein-labeled polyclonal
antibody directed at the target antibody.
Both direct and indirect methods detect viral antigens (e.g.,
cytomegalovirus, herpes simplex virus, and respiratory
viruses) within cultured cells or clinical specimens, and also
Many difficult-to-grow bacterial agents (e.g., Legionella
pneumophila) in clinical specimens.

23. IFAT (Indirect fluorescent antibody test)
An unlabeled (target)
antibody binds a specific
antigen.
The specimen is then
stained with fluorescein-
labeled polyclonal
antibody directed at the
target antibody.

24. Dark-field microscopy
Alters microscopic technique rather than using dye.
Condenser does not allow light to pass directly through the
specimen but at an oblique angle.
Only light that hits objects, such as microorganism in the
specimen will be deflected upward into the objective lens
for visualization.
All other light that passes through the specimen will miss the
objective making the background a dark field.
Used to detect spirochetes

25. Dark-field microscopy
Dark-field microscopy –a) Principle b)Tightly coiled spirochetes
a b

27. a b
Latex agglutination (a) and Coagglutination (b)test

29. ELISA
ELISA plate and well
ELISA machine

30. Sensitivity test
Determination of MIC and MBC
Antibiotic sensitivity testing

31. This test is still
used for the
diagnosis of
fungi,
respiratory
viruses and
arboviruses as
well as to
diagnosis of Q
fever

32. Molecular methods
1. Nucleic acid hybrididization methods
2. PCR based amplification methods
-Multiplex PCR
-Nested PCR
-Real time PCR
3. Non –PCR based amplification methods:
-Nucleic acid sequence based amplification
(NASBA)
- Transcription –mediated amplification (TMA)
-Standard displacement amplification

33. Nucleic acid hybrididization methods
Hybridization methods are based on the ability of two
nucleic acid strands that have complementary base
sequence (homologous) to specifically bond with each other
and form a double stranded molecule or duplex or hybrid.
Hybridization assays require that one nucleic acid strand
(probe) originate from an organism of known identity.

34. Nucleic acid hybrididization methods
Other strand (target) originate from an unknown organism to
be detected.
All hybridization tests must have a means to detect
hybridization.
This is accomplished with the use of a ‘reporter’ molecule
that forms a complex with the single-straded probe DNA.

35. Principles of
nucleic acid
hybridization

36. A) Probe labeled with radioactive reporter-detected by autoradiography
B) Probe labeled with biotin-avidin reporter-detected by colorimetric assay
C) Probe labeled with chemiluminescent reporter—detected by luminometer to detected
emitted light

37. Overview of
PCR

38. Key points for the laboratory diagnosis of
bacteremia/fungemia:
Volume of blood collected, not timing, is most critical.
. A second important determinant is the number of blood culture
sets performed during a given septic episode.
Generally, in adults with a suspicion of BSI, 2–4 blood culture
sets should be obtained in the evaluation of each septic episode.

39. Disinfect the venipuncture site with chlorhexidine or 2%
iodine tincture in adults and children >2 months old
(Chlorhexidine NOT recommended for children <2 months
old).
• Draw blood for culture before initiating antimicrobial
therapy.
• Catheter-drawn blood cultures have a higher risk of
contamination (false positives).

40. Do not submit catheter tips for culture without an
accompanying blood culture obtained by venipuncture.
• Never refrigerate blood prior to incubation.
• Use a 2–3 bottle blood culture set for adults, at least one aerobic
and one anaerobic; use 1–2 aerobic bottles for children.
• Streptococcus pneumoniae and some other gram-positive
organisms may grow best in the anaerobic bottle.

41. Contamination of samples
Blood cultures contaminated with skin flora during
collection are common
Contamination rates should not exceed 3%.
Common blood culture contaminants-
-Coagulase-negative staphylococci,
-Viridians group streptococci
-Diphtheroids,
-Bacillus species other than B.anthracis.

42. Infections Associated With Vascular Catheters
Fundamental to the diagnosis of catheter-associated BSI is
documentation of bacteremia.
The clinical significance of a positive culture from an
indwelling catheter segment or tip in the absence of positive
blood cultures is………….
????????? unknown.
The next essential diagnostic component is demonstrating
that the infection is caused by the catheter.
This usually requires exclusion of other potential primary foci
for the BSI

43. Infections Associated With Vascular Catheters
Documentation of bacteremia.
The clinical significance of a positive culture from an
indwelling catheter segment or tip in the absence of positive
blood cultures is………….
????????? unknown.
Exclusion of other potential primary foci for the BSI

44. Diagnostic techniques for catheter cultures
Time to positivity : 2 hours ahead
Quantitative BCs- 5-fold more
Catheter tip or segment cultures: This method only
detects organisms colonizing the outside of the
catheter, which is rolled onto an agar plate after
which the number of colonies is counted;

45. Central Nervous System (CNS)
Infections
Organisms usually enter the central nervous system by
crossing a mucosal barrier into the bloodstream
followed by
penetration of the blood-brain barrier.
Other routes of infection include:
-direct extension from a contiguous structure
-movement along nerves or
- introduction by foreign devices.

46. Usually 3 or 4 tubes of CSF are collected.
The first tube has the highest potential for contamination with
skin flora
and
should not be sent to the microbiology laboratory for direct
smears, culture, or molecular studies.
A minimum of 0.5–1 mL of CSF should be sent .
Larger volumes (5–10mL) increase the sensitivity of culture and
are required for optimal recovery of mycobacteria and fungi.

47. When the specimen volume is less- prioritization
.
Whenever possible, specimens for culture should be
obtained prior to initiation of antimicrobial therapy.
Serologic diagnosis is based on CSF to serum antibody
index, 4-fold rise in acute to convalescent immunoglobulin
G (IgG) titer, or a single positive immunoglobulin M (IgM).

48. Antibody in CSF-
-CNS infection
-Blood contamination
-Transfer of antibodies across the BBB
Submission of
-acute (3–10 days after onset of symptoms)
-convalescent (2–3 weeks after acute) serum
samples is recommended.

49. Key points for the laboratory diagnosis of CNS
infections:
➢ Whenever possible, collect specimens prior to initiating
antimicrobial therapy.
➢ Two to four blood cultures should also be obtained if
bacterial meningitis is suspected.
➢ Inform the Microbiology Laboratory if unusual organisms
are possible (such as Nocardia, fungi, mycobacteria, etc.),
➢ Do not refrigerate cerebrospinal fluid.
➢ Attempt to collect as much sample as possible (minimum
recommended is 1 mL);
➢ Prioritize multiple test requests on small volume samples.

50. The most common etiologic agents of acute meningitis are:
-Enteroviruses (primarily echoviruses and
coxsackieviruses) and
bacteria-
(Streptococcus pneumoniae and Neisseria meningitidis;
Patient age and other factors (ie, immune status, post
neurosurgery, trauma) are associated with specific bacterial
pathogens.

51. The sensitivity of the Gram stain for the diagnosis of
bacterial meningitis is 60%–80% in patients who have not
received antimicrobial therapy and 40%–60% in patients
who have received treatment.
Bacterial antigen testing on CSF is not recommended but
may have some value in patients who received therapy prior
to specimen collection with negative Gram stain and
negative culture results.
Patients suspected of having bacterial meningitis, at least 2–
4 blood cultures should be performed, but therapy should
not be delayed.

52. Organisms expected to cause chronic meningitis (symptoms
≥4 weeks) include
Mycobacterium tuberculosis
Fungi,
Spirochetes.
Because the sensitivity of nucleic acid amplification
tests (NAAT) for M. tuberculosis in non respiratory
specimens may be poor, culture should also be requested.
The reported sensitivity of culture for diagnosing tuberculous
meningitis is 25%–70% .

53. The highest yields for AFB smear and AFB culture occur
when large volumes (≥5 mL) of CSF are used to perform
the testing.
The cryptococcal antigen test has replaced the India ink
stain for rapid diagnosis of meningitis caused by C.
neoformans or C. gattii.
This test is most sensitive when performed on CSF rather
than serum.

54. Encephalitis
The California Encephalitis Project identified a definite or
probable etiologic agent for only 16% of 1570
immunocompetent patients enrolled from 1998 to 2005
Viral- 69%
Bacterial-20%
Prion-7%
Parasitic-3%
Fungal-1%
A possible cause was identified for an additional 13% of patients
.
Immune status, travel, and other exposure history (insects,
animals,water, sexual) should guide testing.

55. Although the diagnosis of a specific viral cause is usually
based on testing performed on CSF, testing of specimens
collected from other sites may be helpful.
The virus most commonly identified as causing encephalitis
is herpes simplex virus (HSV) with 90% HSV-1.
The sensitivity and specificity of NAAT for HSV
encephalitis are >95%;
HSV is cultured from CSF in <5% of cases .

56. The sensitivity of NAAT performed on CSF for enterovirus
encephalitis is >95% and the
Sensitivity of culture is 65%–75% (recovery from throat or
stool is circumstantial etiologic evidence) .
Because the performance characteristics of molecular testing
for other causes of viral encephalitis are not well established,
serology and repeat molecular testing may be required

57. Central Nervous System Shunt Infections
Shunts are placed to divert cerebrospinal fluid.
The proximal portion is placed in a cerebral ventricle,
intracranial cyst, or the subarachnoid space
. The distal portion may be internalized (peritoneal, vascular,
or pleural space) or externalized.
In total, 5%–15% of shunts become infected . Potential routes
of shunt infection include -
-Contamination at time of placement,
-Contaminationfrom the distal portion (retrograde),
-Breakdown of the skin over the shunt,
-Hematogenous seeding.

58. Blood cultures should also be collected if the shunt
terminates in a vascular space (ventriculo atrial shunt).
Most CNS shunt infections are caused by bacteria.
Fungi are more likely to cause shunt infections in
immunocompromised patients and those receiving total
parenteral nutrition, steroids, or broad-spectrum
antibiotics.

59. Unlike other areas of the diagnostic laboratory, clinical
microbiology is a science of interpretive judgment that is
becomingmore complex, not less.
Even with the advent of laboratory automation and the
integration of genomics and proteomics in microbiology,
interpretation of results still depends on the quality of the
specimens received for analysis.

60. DIAGNOSTIC TECHNOLOGIES
Gram stain/Microscopy
Cultures.
Three major culture media
Enrichment: chocolate and sheep blood
Selective: Thayer-Martin
Differential: MacConkey-ability to ferment lactose

61. 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

62. DIAGNOSTIC TECHNOLOGIES
Immunoserology
Hemagglutination
EIA
Latex agglutination
Compliment fixation
Immunoflorecent

63. 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

64. Molecular Diagnostics
Most widely used is PCR
High sensitivity
High specificity
Diversity
Nucleic acid probes
Do not amplify DNA

65. 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

66. 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 30-60 minutes to
complete

67. Other Uses Of Molecular Diagnostics
Viral load monitoring
Viral genotyping
Bacterial resistance detection
Bacterial genotyping

68. 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

69. Limitation of PCR Technologies
Specimen should be frozen until amplification
No antimicrobial sensitivity is available
Needs the clinician to name the suspect

70. 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

71. Collection and Processing of Clinical
Specimen
Diagnostic Technologies
Culture
Antigen detection
Serology
Molecular diagnostics
Rapid Diagnostic Test
CLIA-Waived tests
Other rapid non-CLIA waived tests

72. Collection And Processing Of Clinical
Specimen
Most important aspect of laboratory medicine
Insufficient quantity
Contamination
Improper transport media
Delay in transportation
Inappropriate storage

73. 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

74. 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

75. 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

76. 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

77. 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: Thayer-Martin
Differential: MacConkey-ability to ferment lactose

78. 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

79. 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