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Laboratory Diagnosis

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Yasmeen Yahya

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Transcript of Laboratory Diagnosis

Laboratory Diagnosis
Chapter 9
AP. Dr. Said S. AlGhora
CHAPTER CONTENTS
Approach to Laboratory Diagnosis.
Bacteriologic Methods.
Blood Cultures.
Throat Cultures.
Sputum Cultures.
Spinal Fluid Cultures.
Stool Cultures.
Urine Cultures.
Genital Tract Cultures.
Wound & Abscess Cultures.
Immunologic Methods.
Identification of an Organism with Known Antiserum.
Identification of Serum Antibodies with Known Antigens.
Nucleic Acid-Based Methods.
APPROACH TO LABORATORY DIAGNOSIS
The laboratory diagnosis of infectious diseases involves
two main approaches:
one is the
immunologic (serologic)
approach, in which the organism is identified by detection of
antibodies
against the organism in the patient's serum , and the other is the
bacteriologic
approach, in which the organism is identified by
staining and culturing
the organism.
Bacteriologic Approach
In the bacteriologic approach to the diagnosis of infectious diseases, several important
steps
precede the actual laboratory work, namely,
(1) choosing the appropriate specimen to examine.
(2) obtaining the specimen properly to avoid contamination from the normal flora.
(3) transporting the specimen promptly to the laboratory or storing it correctly.
(4) providing essential information to guide the laboratory personnel.
(3) Identifying the organism
In general, there are three approaches to the bacteriologic laboratory work:
(1) Observing the organism in the microscope after staining.
(2) Obtaining a
pure culture
of the organism by inoculating it onto a bacteriologic medium.
by using biochemical reactions, growth on selective media, DNA probes, or specific antibody reactions. Which of these approaches are used and in what sequence depend on the type of specimen and organism.
After the organism is grown in pure culture, sensitivity to various antibiotics is determined by various procedures.
A general approach to the diagnosis of a bacterial infection:
This approach emphasizes the importance of performing a Gram stain and obtaining a “pure culture” of the organism. However, sometimes the organism is
not
recovered by culturing, and other techniques must be used.
Some approaches to making a diagnosis when the cultures are
negative:

One approach that is commonly used is
serologic testing
, which determines the presence of
antibodies specific for the organism
. In most cases, a
fourfold rise in antibody titer
between the acute- and convalescent-phase serum samples is considered to be
significant
.
The concentration of antibodies circulating in the bloodstream of an individual. A
rising titer
usually indicates the body's response to antigens associated with an
active infection
.
Antibody Titer
TO
Obtaining a pure culture involves culturing the organism on bacteriologic agar.
Bacterial Culture
A
pure culture
is a laboratory culture that contains a
single species
of organism. A pure culture is usually derived from a mixed culture. A
mixed culture
is a culture that contains
many species
.
Pure vs. Mixed Culture
Agar
is a jelly-like substance, obtained from algae. It is used to provide a

growth medium
using a mix of agar and other nutrients in which microorganisms, including bacteria and fungi, can be cultured and observed under the microscope. Agar is
indigestible
for many organisms so that microbial growth does not affect the gel used and it remains stable.
Don't worry,, each one will described at its suitable time..
Selective Vs. Differential Media
Selective media

allow certain types of organisms to grow, and inhibit the growth of other organisms.
The selectivity is accomplished in several ways. For example, organisms that can utilize a given sugar are easily screened by making that sugar the only carbon source in the medium.

Differential media

contain other compounds that allow one type of bacteria to be differentiated
from another based on some biochemical reaction. Owing to the presence of certain dyes or chemicals in the media, the organisms will produce characteristic changes or growth patterns that are used for identification or differentiation.

Blood agar contains inhibitors for certain bacteria, such as members of the
Neisseria
and
Haemophilus genera
, and the blood must be heated to inactivate these inhibitors.
These bacteria therefore are grown on cooked blood agar or
chocolate agar

(so named because the heated blood turns a chocolate color).

Other media contain either specific growth factors required for the bacteria to grow or contain antibiotics that inhibit normal flora, which allows the pathogenic bacteria to obtain sufficient nutrients to grow.
Initially,
blood agar
is used because it supports the growth of many bacteria and the type of
hemolysis
can be observed.
Blood agar contains
red blood cells
, but it should be noted that
viruses and obligate intracellular bacteria
, such as
Chlamydia

and

Rickettsia
, will
not grow
on blood agar. Red blood cells do not have a functioning nucleus and therefore are incapable of supporting the growth of either viruses or the obligate intracellular bacteria.
BACTERIOLOGIC METHODS
Blood
Cultures
Blood cultures are performed most often when
sepsis, endocarditis, osteomyelitis, meningitis,
or
pneumonia
is suspected. The organisms most frequently isolated from blood cultures are two gram-positive cocci,
Staphylococcus aureus
and
Streptococcus pneumoniae
, and three gram negative rods,
Escherichia coli
,
Klebsiella pneumoniae
,

and
Pseudomonas aeruginosa
.
It is important to obtain at least three 10-mL blood samples in a 24-hour period because the number of organisms can be small and their presence intermittent. The site for venipuncture must be cleansed with 2%
iodine
to prevent contamination by members of the flora of the skin, usually
Staphylococcus epidermidis.

The blood obtained is added to 100 mL of a rich growth medium such as
brain– heart infusion broth.


Whether one or two bottles are inoculated varies among hospitals. If two bottles are used, one is kept under
anaerobic

conditions and the other is not. If one bottle is used, the low oxygen tension at the bottom of the bottle permits anaerobes to grow.
Blood cultures are checked for
turbidity
or for
CO2 production
daily for 7 days or longer. If growth occurs, Gram stain, subculture, and antibiotic sensitivity tests are performed.

If no growth is observed after 1 or 2 days, blind subculturing onto other media may reveal organisms. Cultures should be held for 14 days when
infective endocarditis, fungemia
, or infection by
slow-growing bacteria
(e.g.,
Brucella
) is suspected.
Throat
Cultures
Throat cultures are used primarily to detect the presence of
group A-hemolytic streptococci
(
Streptococcus pyogenes
), an important and treatable cause of
pharyngitis
. They are also used when
diphtheria
,
gonococcal pharyngitis
, or
thrush ( Candida )
is suspected.
When the specimen is being obtained, the swab should touch not only the
posterior pharynx
, but also
both tonsils
or
tonsillar fossae
as well. The material on the swab is inoculated onto a

blood agar plate
and streaked to obtain single colonies.



Note that a

Gram stain
is typically
not done
on a throat swab because it is impossible to distinguish between the appearance of the normal flora streptococci and
Str. pyogenes.
If colonies ofβ
B-hemolytic streptococci
are found after 24 hours of incubation at 35°C, a
bacitracin
disk is used to determine whether the organism is likely to be a group
A streptococcus.
If growth is
inhibited
around the disk,

it is a group A streptococcus; if not, it is a non group A B-hemolytic streptococcus.
Bacitracin-resistant (no zone of inhibition).
Streptococcus agalactiae growing around Bacitracin Differentiation Disk. Incubated in CO2 on Blood Agar for 24 hours at 35ºC.

Bacitracin-sensitive (zone of inhibition).
Streptococcus pyogenes inhibition zone around Bacitracin Differentiation Disk. Incubated in CO2 on Blood Agar for 24 hours at 35ºC.
Sputum
Cultures
Sputum cultures are performed primarily when
pneumonia
or
tuberculosis

is suspected. The most frequent cause of
community-acquired pneumonia
is
Str. pneumoniae
, whereas
Sta. aureus
and gram-negative rods, such as
K. pneumoniae
and
P. aeruginosa
, are common causes of
hospital-acquired pneumonias.

It is important that the specimen for culture really be sputum,
not saliva.
Examination of a gram-stained smear of the specimen frequently reveals whether the specimen is satisfactory. A reliable specimen has more than
25 leukocytes
and fewer than
10 epithelial cells
per 100× field.
An unreliable sample can be misleading and should be
rejected
by the laboratory.

Culture of the sputum on
blood agar
frequently reveals characteristic colonies, and identification is made by various
serologic
or
biochemical tests.

Cultures of
Mycoplasma
are infrequently done; diagnosis is usually confirmed by a rise in
antibody titer
.

If
Legionella pneumonia
is suspected, the organism can be cultured on
charcoal-yeast agar
, which contains the high concentrations of iron and sulfur required for growth.
If

tuberculosis
is suspected, an
acid-fast stain
should be done immediately and the sputum cultured on special media, which are incubated for at least
6 weeks.
In diagnosing aspiration pneumonia and lung abscesses,
anaerobic cultures
are important.
Spinal Fluid
Cultures
Spinal fluid cultures are performed primarily when
meningitis
is suspected. Spinal fluid specimens from cases of
encephalitis, brain abscess,
and
subdural empyema
usually show
negative
cultures. The most important causes of acute bacterial meningitis are three
encapsulated
organisms:
Neisseria meningitidis, Str. pneumoniae,
and
Haemophilus influenzae.
Because acute meningitis is a medical
emergency
, the specimen should be taken immediately to the laboratory. The gram-stained smear of the sediment of the centrifuged sample guides the immediate empirical treatment.

If organisms resembling
N. meningitidis, H. influenzae,

or
Str. Pneumoniae
are seen, the
quellung
test or immunofluorescence with specific antisera can identify the organism rapidly.
In cases of
subacute meningitis
,
Mycobacterium tuberculosis
and the fungus
Cryptococcus neoformans
are the most common organisms isolated.
Acid-fast stains
of the spinal fluid should be performed, although
M. tuberculosis
may not be seen, because it can be present in small numbers. The fluid should be cultured and the cultures held for a minimum of
6 weeks
.
C. neoformans
, a budding yeast with a
prominent capsule
, can be seen in spinal fluid when
India ink
is used.
Immunologic tests
to detect the presence of capsular antigen in the spinal fluid can be used to identify
N. meningitidis
,
Str. pneumoniae, H. influenzae, group B streptococci, E. coli,
and
C. neoformans.
The two tests most frequently used are
latex particle agglutination
and
counterimmunoelectrophoresis.
Stool
Cultures
Stool cultures are performed primarily for cases of
enterocolitis
. The most common bacterial pathogens causing diarrhea in the United States are
Shigella, Salmonella,
and
Campylobacter
.
E. coli O157
strains are also an important cause of diarrhea.
A direct
microscopic examination
of the stool can be informative from two points of view:

(1) A
methylene blue stain
that reveals many
leukocytes
indicates that an
invasive
organism rather than a toxigenic one is involved.

(2) A
Gram stain
may reveal large numbers of certain organisms, such as
staphylococci, clostridia,
or
campylobacters
.
Gram stain of the stool is not usually done because the large numbers of bacteria in the
normal flora
of the colon make the interpretation difficult.

For culture of
Salmonella
and
Shigella
, a selective, differential medium such as
MacConkey
or
Eosin methylene blue (EMB)
agar is used. These media are
selective
because they allow
gram-negative
rods to grow but inhibit many gram positive organisms. Their
differential
properties are based on the fact that
Salmonella
and
Shigella

do not ferment lactose
, whereas many other enteric gram-negative rods do. If non– lactose-fermenting colonies are found, a
triple sugar iron (TSI)
agar slant is used to distinguish
Salmonella
from
Shigella.
Some species of
Proteus
resemble
Salmonella
on
TSI
agar but can be distinguished because they produce the enzyme
urease
, whereas Salmonella does not. The organism is further identified as either a
Salmonella
or
a Shigella
species by using a
specific antisera
to the organism's cell wall
O antigen
in an agglutination test. This is usually done in hospital laboratories, but precise identification of the species is performed in public health laboratories.
Campylobacter jejuni
is cultured on a
ntibiotic-containing media
(e.g.,
Skirrow's agar
) at 42°C in an atmosphere containing 5% O 2 and 10% CO 2 . It grows well under these conditions, unlike many other intestinal pathogens. Although the techniques are available, stool cultures are infrequently performed for organisms such as
Yersinia enterocolitica
,
Vibrio parahaemolyticus
, and
enteropathic
or
toxigenic E. coli.
Despite the presence of large numbers of anaerobes in feces, they are rarely pathogens in the intestinal tract, and anaerobic cultures of stool specimens are therefore unnecessary.
Stool specimens that are grossly
bloody
are typically cultured on
MacConkey-sorbitol media
.
E. coli O157
strains do
not
ferment sorbitol and appear as
colorless
colonies, whereas typical
E. coli
strains do ferment sorbitol and appear
red
.
Urine
Cultures
Urine cultures are performed primarily when
pyelonephritis
or
cystitis
is suspected. By far the
most
frequent cause of urinary tract infections is
E. coli.
Other common agents are
Enterobacter, Proteus,
and
Enterococcus faecalis.

It is commonly accepted that a bacterial count of at least 100,000/mL must be found to conclude that significant bacteriuria is present (in asymptomatic persons). There is evidence that as few as 100/mL are significant in symptomatic patients. For this determination to be made, quantitative or semiquantitative cultures must be performed. There are several techniques:
Urine in the bladder of a healthy person is
sterile
, but it acquires organisms of the normal flora as it passes through the distal portion of the urethra. To avoid these organisms, a
midstream specimen
, voided after washing the external orifice, is used for urine cultures. In special situations,
suprapubic aspiration
or
catheterization
may be required to obtain a specimen. Because urine is a good culture medium, it is essential that the cultures be done within
1 hour
after collection or stored in a refrigerator at 4°C for no more than 18 hours.

Genital Tract
Cultures
Genital tract cultures are performed primarily on specimens from individuals with an
abnormal discharge
or on specimens from asymptomatic contacts of a person with a
sexually transmitted disease
. One of the most important pathogens in the genital tract is
Neisseria gonorrhoeae
. The laboratory diagnosis of gonorrhea is made by microscopic examination of a gram-stained smear and by culture of the organism.
Specimens are obtained by
swabbing
the urethral canal (for men), the cervix (for women), or the anal canal (for men and women). A urethral discharge from the penis is frequently used. Because
N. gonorrhoeae
is very delicate, the specimen should be inoculated directly onto a
Thayer- Martin chocolate agar
plate or onto a special transport medium (e.g., Trans-grow).
Gram-negative diplococci found intracellularly
within neutrophils
on a smear of a urethral discharge from a man have over 90% probability of being
N. gonorrhoeae
. Because smears are less reliable when made from swabs of the endocervix and anal canal, cultures are necessary. The finding of only extracellular diplococci suggests that these
neisseriae
may be members of the normal flora and that the patient may have
nongonococcal urethritis.
Nongonococcal urethritis and cervicitis are also extremely common infections. The most frequent cause is
Chlamydia trachomatis
, which cannot grow on artificial medium but must be grown
in living cells.
For this purpose, cultures of human cells or the yolk sacs of embryonated eggs are used. The finding of typical
intracytoplasmic inclusions
when using
Giemsa stain
or
fluorescent antibody
is diagnostic. Because of the difficulty of culturing
C. trachomatis,
nonbacteriologic methods, such as
enzyme-linked immunosorbent assay (ELISA)
to detect chlamydial antigens in exudates or urine or DNA probe assays to detect chlamydial nucleic acids, are now often used to diagnose sexually transmitted diseases caused by this organism.
Because
Treponema pallidum
, the agent of
syphilis
, cannot be cultured, diagnosis is made by microscopy and serology. The presence of
motile spirochetes
with typical morphologic features seen by dark field microscopy of the fluid from a painless genital lesion is sufficient for the diagnosis.
The
serologic
tests fall into two groups:
(1) the
nontreponemal antibody
tests such as the Venereal Disease Research Laboratory
(VDRL)
or rapid plasma reagin
(RPR)
test.

(2) the
treponemal antibody
tests such as the fluorescent treponemal antibody-absorption
(FTA-ABS)
test.

Wound & Abscess
Cultures
A great variety of organisms are involved in wound and abscess infections. The bacteria most frequently isolated differ according to anatomic site and predisposing factors.

Abscesses of the
brain, lungs,
and
abdomen
are frequently caused by
anaerobes
such as
Bacteroides fragilis
and gram positive cocci such as
Sta. aureus
and
Str. pyogenes.

Traumatic open-wound infections are caused primarily by members of the
soil flora
such as
Clostridium perfringens
; surgical-wound infections are usually due to
Sta. aureus
. Infections of dog or cat bites are commonly due to
Pasteurella multocida
, whereas human bites primarily involve the mouth anaerobes.

Because anaerobes are frequently involved in these types of infection, it is important to place the specimen in anaerobic collection tubes and transport it promptly to the laboratory. Because many of these infections are due to
multiple organisms
, including mixtures of anaerobes and nonanaerobes, it is important to culture the specimen on
several different media
under different atmospheric conditions. The Gram stain can provide valuable information regarding the range of organisms under consideration.
If the patient
cannot cough
and the need for a microbiologic diagnosis is strong,
induction of sputum, transtracheal aspirate, bronchial lavage,
or
lung biopsy
may be necessary. Because these procedures bypass the normal flora of the upper airway, they are more likely to provide an accurate microbiologic diagnosis. A preliminary assessment of the cause of the pneumonia can be made by
Gram stain
if large numbers of typical organisms are seen.
Legionella pneumophila colonies on Buffered Charcoal Yeast Extract Agar
Cultures are done on
blood
and on
chocolate agar
and incubated at 35°C in a 5% CO2 atmosphere.
Hematin
and nicotinamide adenine dinucleotide
(NAD)
(
factors X
and
V
, respectively) are added to enhance the growth of H. influenzae.
Latex Particle Agglutination
Counterimmunoelectrophoresis
Counter\ Immuno\ electrophoresis
MacConkey agar with lactose fermenters and non lactose fermenters colonies
Escherichia coli colonies growing on EMB Agar. Incubated aerobically for 24 hours at 35°C.
Salmonella enterica colonies growing on EMB Agar. Incubated aerobically for 24 hours at 35°C.
EMB Agar
TSI Agar
Where Have I heard this before?
"5% O2 and 10% CO2 conditions"
Microaerophilic Growth
Skirrow's Agar
(1) A
calibrated loop
that holds 0.001 mL of urine can be used to streak the culture.
(2) serial 10-fold dilutions can be made and samples from the dilutions streaked.
(3) a screening procedure suitable for the physician's office involves an agar-covered
“paddle”
that is dipped into the urine-after the paddle is incubated, the density of the colonies is compared with standard charts to obtain an estimate of the concentration of bacteria.
Chlamydia
I
ntracytoplasmic Inclusions
You've passed half of the way,,

Have a break..
IMMUNOLOGIC METHODS
There are essentially two basic approaches:
Identification of an Organism with Known Antiserum
Identification of Serum Antibodies with Known Antigens
Capsular Swelling (Quellung) Reaction
Several bacteria can be identified directly in clinical specimens by this reaction, which is based on the microscopic observation that the capsule swells in the presence of homologous antiserum. Antisera against the following organisms are available: all serotypes of
Str. Pneumoniae
(Omniserum),
H. influenzae type b
, and
N. meningitidis groups A and C
.
Slide Agglutination Test
Antisera can be used to identify
Salmonella
and
Shigella
by causing agglutination (clumping) of the unknown organism.
Antisera directed against the
cell wall O antigens
of
Salmonella
and
Shigella
are commonly used in hospital laboratories. Antisera against the
flagellar H antigens
and the
capsular Vi antigen
of
Salmonella
are used in public health laboratories for epidemiologic purposes.
Latex Agglutination Test
Latex beads coated with specific antibody are agglutinated in the presence of the homologous bacteria or antigen. This test is used to determine the presence of the
capsular antigen
of
H. influenzae
,
N. meningitidis
, several species of
streptococci
, and the yeast
C. neoformans
.
Counterimmunoelectrophoresis Test
In this test, the
unknown bacterial antigen
and a
known specific antibody
move toward each other in an
electrical field
. If they are homologous, a precipitate forms within the agar matrix. Because
antibodies
are
positively
charged at the pH of the test, only
negatively
charged
antigens
, usually capsular polysaccharides, can be assayed. The test can be used to detect the presence in the spinal fluid of the capsular antigens of
H. influenzae, N. meningitidis, Str. pneumoniae,
and
group B streptococci.
Enzyme-Linked Immunosorbent Assay
(ELISA)
In this test, a specific antibody to which an easily assayed enzyme has been linked is used to detect the presence of the homologous antigen.
This test is useful in detecting a wide variety of
bacterial, viral,
and
fungal infections
.
Fluorescent Antibody Tests
A variety of bacteria can be identified by exposure to known
antibody labeled with fluorescent dye
, which is detected visually in the ultraviolet microscope. Various methods can be used, such as the direct and indirect techniques.
Slide or Tube Agglutination Test
In this test,
serial two-fold dilutions
of a sample of the patient's serum are mixed with standard bacterial suspensions.
The
highest dilution
of serum capable of agglutinating the bacteria is the
titer of the antibody
. As with most tests of a patient's antibody, at least a
four-fold rise
in titer between the early and late samples must be demonstrated for a diagnosis to be made. This test is used primarily to aid in the diagnosis of

typhoid fever, brucellosis, tularemia, plague, leptospirosis,
and
rickettsial diseases.
Serologic Tests for Syphilis
The detection of antibody in the patient's serum is frequently used to diagnose syphilis, because T. pallidum does not grow on laboratory media.
There are
two kinds of tests:
(1) The nontreponemal tests
They use a
cardiolipinlecithin– cholesterol mixture
as the antigen, not an antigen of the organism. Cardiolipin (diphosphatidylglycerol) is a lipid extracted from normal beef heart. Flocculation (clumping) of the cardiolipin occurs in the presence of antibody to
T. pallidum
. The
VDRL
and
RPR
tests are nontreponemal tests commonly used as screening procedures. They are
not specific for syphilis
but are inexpensive and easy to perform.
(2) The treponemal tests
They use

T. pallidum
as the
antigen
. The two most widely used treponemal tests are the
FTA-ABS
and the
MHA-TP
(microhemagglutination–
Treponema pallidum
) tests.
Cold Agglutinin Test
Patients with
Mycoplasma pneumoniae
infections develop

autoimmune antibodies

that agglutinate human red blood cells in the cold
(4°C)
but not at 37°C. These antibodies occur in certain diseases other than
Mycoplasma
infections; thus false-positive results can occur.
In the FTA-ABS test, the patient's serum sample, which has been absorbed with
treponemes other than
T. pallidum
to remove nonspecific antibodies, is reacted with nonviable
T. pallidum
on a slide. Fluoresceinlabeled antibody against human
immunoglobulin G (IgG)
is then used to determine whether IgG antibody against T. pallidum is bound to the organism.
FTA-ABS test
In the MHA-TP test, the patient's serum sample is reacted with
sheep erythrocytes
coated with antigens of
T. pallidum
. If antibodies are present,
hemagglutination
occurs.
MHA-TP test
NUCLEIC ACID BASED METHODS
There are
three
types of nucleic acid–based tests used in the diagnosis of bacterial diseases:
nucleic acid amplification tests
,
nucleic acid probes
, and

nucleic acid sequence analysis.
Nucleic acid–based tests are highly
specific, quite sensitive
(especially the amplification tests), and much faster than culturing the organism. These tests are especially useful for those bacteria that are difficult to culture, such as
Chlamydia
and
Mycobacterium
species.
Nucleic acid amplification tests
utilize the polymerase chain reaction (PCR) or other amplifying process to increase the number of bacteria-specific DNA or RNA molecules so the
sensitivity
of the test is significantly
higher
than that of unamplified tests. Many bacteria can be identified using these tests, but they are especially useful in detecting
Chlamydia trachomatis
and
Neisseria gonorrhoeae
in urine samples in STD clinics.
Tests that use
nucleic acid probes
are designed to detect bacterial DNA or RNA directly
(without amplification)
using a labeled DNA or RNA probe that will hybridize specifically to the bacterial nucleic acid. These tests are
simpler
to perform than the amplification tests but are
less sensitive.
Nucleic acid sequence analysis
is used to identify bacteria based on the base sequence of the organism's ribosomal RNA. An organism that has never been cultured,
Tropheryma whipplei
, was identified using this approach.
Thank
you
With Regards:
Reem A. AlGazali
Yasmeen Y. Sarraj
Self Assessment Questions

When a substance is added to a solid medium which inhibits the growth of unwanted bacteria but permits the growth of wanted bacteria, it is known as:

A. differential medium
B. enriched medium
C. enrichment medium
D. selective medium

The medium which allows the growth of more than one microorganisms of interest but with morphologically distinguishable colonies is known as:

A. selective medium.
B. enrichment medium.
C. differential medium.
D. none of these.
Answer:
Option D

Answer:
Option C
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