Download Infectious diseases

INFECTIOUS DISEASES
Infectious diseases
 infectious diseases are distinctive enough to be
identified clinically.
 Most pathogens, however, can cause a wide
spectrum of clinical syndromes in humans.
Conversely, a single clinical syndrome may result
from infection with any one of many pathogens.
Influenza virus infection, for example,
causes a wide variety of respiratory syndromes
that cannot be distinguished clinically from
those caused by streptococci, mycoplasmas, or
more than 100 other viruses.
1.
It is necessary to use
microbiological
laboratory methods
to identify a specific
etiologic agent.
2. Diagnostic medical microbiology is the discipline that
job of
the clinical microbiology laboratory
identifies etiologic agents of disease. The
is to test specimens from patients for microorganisms
that are, or may be, a cause of the illness and to
provide information (when appropriate) about the in
vitro activity of antimicrobial drugs against the
microorganisms identified
 The staff of a clinical microbiology laboratory
should be qualified to advise the physician as well as
process specimens.
 The
physician
should
supply
salient
information about the patient, such as age and
sex, tentative diagnosis or details of the clinical
syndrome, date of onset, significant exposures,
prior antibiotic therapy, immunologic status, and
underlying conditions.
 The clinical microbiologist participates in decisions
regarding the microbiologic diagnostic studies to be
performed, the type and timing of specimens to be
collected, and the conditions for their
transportation and storage. Above all, the clinical
microbiology laboratory, whenever appropriate,
should provide an interpretation of laboratory
results.
Manifestation of Infection
An indication of the existence, presence of something, especially an
illness.)
(
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The manifestations of an infection depend on many factors, including
 the
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site of acquisition or entry of the
microorganism;
organ or system tropisms of the microorganism;
microbial virulence;
the age, sex, and immunologic status of the
patient; underlying diseases or conditions;
and the presence of implanted prosthetic
devices or materials.
The signs and symptoms of infection may be localized, or they may be systemic, with
fever, chills, and hypotension. In some instances the manifestations of an
infection are sufficiently characteristic to suggest the diagnosis; however, they
are often nonspecific.
Microbial Causes of Infection
 Infections may be caused by bacteria (including mycobacteria, chlamydiae,
mycoplasmas, and rickettsiae), viruses, fungi, or parasites.

Infection may be endogenous or exogenous.
 In endogenous infections, the microorganism (usually a bacterium) is a
component of the patient;s indigenous flora. Endogenous infections can
occur when the microorganism is aspirated from the upper to the lower
respiratory tract or when it penetrates the skin or mucosal barrier as a
result of trauma or surgery.
 In contrast, in exogenous infections, the microorganism is acquired from
the environment (e.g., from soil or water) or from another person or an
animal.
important to establish the cause of an
infection, the differential diagnosis is based on a careful
1.
Although it is
2.
history, physical examination, and appropriate radiographic and laboratory studies,
including the selection of appropriate specimens for microbiologic examination.
Results of the history, physical examination, and radiographic and laboratory studies
allow the physician to request tests for the microorganisms most likely to be the
cause of the infection.
SAMPLE COLLECTION,
PROCESSING AND STORAGE
Specimen Selection, Collection
and Processing
 Specimens selected for microbiological examination
should reflect the disease process and be collected in
sufficient quantity to allow complete microbiologic
examination.
 The number of microorganisms per milliliter of a body
fluid or per gram of tissue is highly variable,
 Swabs, although popular for specimen collection,
frequently yield too small a specimen for accurate
microbiologic examination and should be used only to
collect material from the skin and mucous
membranes.
Specimen Selection, Collection and
Processing cont……..
contamination
 Because skin and mucous membranes have a large and
diverse indigenous flora, every effort must be made to
minimize specimen contamination during collection.
 Contamination may be avoided by various means. The
skin can be disinfected before aspirating or incising a
lesion. Alternatively, the contaminated area may be
bypassed altogether. Examples of such approaches are
 transtracheal puncture with aspiration of lower
respiratory secretions or suprapubic bladder puncture
with aspiration of urine. It is often impossible to
collect an uncontaminated specimen, and
decontamination procedures, cultures on selective
media, or quantitative cultures must be used.
Specimen Selection, Collection and
Processing cont……..
 If possible, specimens should be collected
before the
antibiotics.
administration
of
Above all, close communication between the
clinician and the microbiologist is
essential to ensure that appropriate specimens
are selected and collected and that they are
appropriately examined.
Specimen Selection, Collection and
Processing cont……..
 Sample Collection, such as handling, labeling,
processing, aliquoting, storage, and
transportation, may affect the results of the
study
 If case sample are handled differently from
controls samples, differential
misclassification may occur
Information linked to Sample
 Time and date of collection
 Recent diet and supplement use,
 current medication use
 History of any disease
 Recent medical illness
 Storage conditions
Quality Assurance
 Adoption of standardized operation
procedures (SOP) for each aspect of
biospecimen handling
 Stored specimens should be tested on a
regular basis
 Aliquoting material into multiple small
vials
Quality Assurance
 Storing
 Storing each person’s specimen in at least two
different physical locations to avoid the
likelihood of loss of a large volume of specimen as a
result of accidental thawing due to freezer failure or
electronic blackout.
Storage
It is critical to maintain careful records of
the identity and location of all materials,
with
particular attention to storage history,
occurrence of temperature fluctuation
monitoring of stored control specimen in
order to check the effects of storage
duration.
Storage
Samples stored on the top of the freezer may
be exposed to more extreme temperature
fluctuation then those stored at the bottom.
Timing
 For studies of hormones, which have
 hourly,
 daily and
 monthly timing of sample collection is critical.
 It is critical to obtain information at the time
of specimen collection, e.g.,
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time and date of draw,
volumes and type of specimen,
medical illness,
medication use etc…….
Processing
 The sample processing depends on the
marker needed. Investigator must design
studies to fit the requirement of the critical
biomarkers.
Types of Biospecimens
individuals who already diagnosed
 Collection of biospecimens from individuals
who already diagnosed as having illness to
characterize the history of the disease.
Many collections of tissues including tumor
and normal tissues. It will be much better if
the related epidemiological data are
also collected.
Types of Biospecimens: Blood
skilled technicians
 The use of skilled technicians and precise
procedures when perform phlebotomy are
important because painful, prolonged or repeated
attempts at venepuncture can cause patient
discomfort or injury and result in less than
optimum quality or quantity of sample.
Types of Biospecimens: Blood
 Plasma
 Serum
 Lymphocytes
 Erythrocytes
 Platelets
Blood Sample Collection
 When a large amount of blood sample needed, an
evacuated tube system with interchangeable glass
tubes can be used to avoid multiple
venepunctures.
 Evacuated tubes are commercially prepared with or
without additives and with sufficient vacuum to
draw a predetermined blood volume per tube.
Sterile Blood needles; Sterile Syringes; Plain Vacutainer; Blood
Tubes; Alcohol Prep Pads;
Tourniquet
Blood Collection: Color-code Tubes
Red-top tubes contain no additives. These tubes are used for tests
performed
on serum samples and
DNA.
 When you use the red-top tubes,
the sample can be placed for 1-2
hours so that the serum and
blood clots will be separated.
Blood clots can be
used for DNA analysis.
Blood Collection: Color-code
Tubes
 Lavender-top tubes contain EDTA, commonly used
clinically for complete blood cell counts.
 This is the way to obtain
 for DNA extraction,
 plasma Biochemistry
 red blood cells for other assays.
EDTA
EDTA is a anticoagulant. It works by calcium
chelation and is used clinically in heamatology
studies. It is well suited
 to DNA-based assays,
 but has problems for cytogenetic analysis.
Blood Collection: Color-code
Tubes
 Green-top tubes contain heparin
 Heparin is an anticoagulant. There are
some reports of problems with heparin
in PCR assays, studies generally find
that there are no major difference in the
use of EDTA or heparin
Blood Collection: Color-code
Tubes cont….
 Blue-top tubes contain sodium citrate and
citric acid
 Citrate also works by calcium chelation and is used
in
 coagulation studies and
 blood banking.
Blood Collection: Color-code
Tubes cont….
 Black-top tubes contain sodium oxalate
Blood Collection: Color-code
Tubes cont….
 Yellow-top tubes contain acid-citrate-
dextrose (ACD) solution.
Blood Collection: Color-code
Tubes cont….
 Grey-top tubes contain a glycolytic
inhibitor.
Dried Blood Spot
Dried blood spot specimens:
 Small quantities of blood adequate for the
characterization of DNA.
 Not require venepuncture or low temperature
condition during collection, processing and
storage
 Can be from whole blood or antocoagulated
with EDTA
Dried Blood Spot
 Blood specimen is spotted onto clean slides
or paper or cotton cloth.
 Transported and stored at room temperature
 Serves as a good source of high-molecular-
weight DNA
 A quantity of 50 ul of dried blood can provide
0.5 ug DNA, sufficient for multiple PCR-based
assays
Processing
Serum fatty acids should be measured
 within 2 weeks at 4 degree C,
within a few months at –20 degree C,
and
 within a year at –80 degree C
Urine Collection
Urine is an ultrafiltrate of the plasma. It
can be used
to evaluate and monitor body metabolic
disease process,
exposure to xenobiotic agents,
Urine Collection

Urine collection is not readily
obtainable. However, it is more
inconvenient than blood collection.
The type of urine selected and the
collection procedure used to
depend on the tests to be performed.
Urine Collection
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First morning
Random
Fractional
timed
Urine Collection
 Clean and dry plastic or glass containers (50
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3000 ml capacity)
A preservative may be needed depending on
the proposed assay
Total volume must be recorded
The specimen well mixed to ensure
homogeneity
Aliquots for specific assays
Urine Collection
 Time frame for measurement of proposed
assay.
 Microbial contamination
 Cost of storing large volumes of material
 effect of long-term storage on quantitative or
qualitative detection
Tissue Collections
 Confirming clinical diagnosis by
histological analysis
 Examining tumor characteristics at
chromosome and molecular level
Tissue Collections
 It requires to collect
more materials
than it is necessary for pathological evaluation
 When possible, the tissue sample should
contain both tumor and normal
tissues to permit to study different
characteristics of the two tissues.
Tissue Collections
 Formalin-fixed paraffin-embedded tissue
specimens
 The tissue is embedded in frozen section
support media and stored at –70 degree C
 Snap frozen tissues.
Hair
 Easy available biological tissue whose typical
morphology may reflect disease conditions
within the body
 Provides permanent record of trace elements
associated with normal and abnormal
metabolism
 A source for occupational and environmental
exposure to toxic metals
Hair
Hair analysis provides long-term information
from months to years, concerning both the
severity and pattern of drug use.
 Hair roots can be optimal source of DNA for
PCR analysis and
 permit easy collection,
 transportation and
 low overall costs.
Nail Clippings
 Toenail or fingernail clippings are obtained in
a very easy and comfortable way.
 They do not require
 processing,
 storage and
 shipping condition and thus suitable
for large epidemiological studies
Nail Clippings
 Trace elements
 Selenium levels
 Arsenic levels
 Less likely to be contaminated by
environmental factors
Saliva
 It is an efficient, painless and relatively
inexpensive source of biological materials for
certain assays
 It provides a useful tool for measuring
endogenous and xenobiotic compounds
Saliva Measurements
 Corticosteroids
 Antibodies to HIV-1
Feaces
 Certain cells of interest
 Infectious markers
 Oncogenes
Shipping
 Sample shipping requirements depends on the
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time,
distance,
climate,
season,
method of transport,
type of specimen and markers to be assayed.
 Polyurethane boxes containing dye ice are used to
ship and transport samples that require low
temperature. For samples require very low
temperature, liquid nitrogen container can be
used. The quantity of dry ice should be carefully
calculated, based on estimated time of trip.
Safety
 Protect specimen from contamination
 Workers safety, HIV, HBV
Procedures
 Standardized approaches in order to ensure
quality control
 Biological specimen collection manual
 Manuals for field trip preparation, packing
and shipping samples
 Protocols for lab assays