Interesting Cases in Clinical
Microbiology
Lisa H. Hochstein, MS, MLS (ASCP)CM
Associate Professor/Program Director,
Clinical Laboratory Sciences
St. John’s University
Queens, NY
Objectives
• Discuss rarely seen pathogens isolated in a
clinical microbiology laboratory
• Correlate laboratory data, patient symptoms
and travel history to correctly identify these
rarely seen pathogens
• Discuss the need for communication between
the physicians and the clinical microbiology
laboratory
Case #1
• 20 month old female who was admitted to a
community hospital
• Patient had arrived 4 days earlier from
Pakistan
• Patient’s chief complaint was diarrhea and
vomiting for 6 days duration
• Initial cultures sent to the microbiology lab
included blood, stool and urine
Case #1
• Also sent to microbiology was stool for ova
and parasites and rotavirus
• A complete blood count (CBC) including
differential, urinalysis and a basic metabolic
panel were also sent for analysis
Case #1
• Cell profile results are listed below:
WBC count – 21,600/mm3
RBC count – 3.75M/mm3
Hemoglobin – 6.6 gm/dL
Hematocrit – 27.1%
Differential indicated an increase of
segmented neutrophils and bands
Case #1
• Basic metabolic panel results showed:
decreased levels of Na, K, Ca and CO2
• Urinalysis showed results within normal
parameters with a trace of protein and
bacteria
• Based on these lab results, the patient was
started on IV fluids for dehydration, given iron
syrup for low blood hemoglobin and Bcomplex and lysine for lack of appetite
Case #1
• Microbiology results showed:
Blood culture drawn on admission – no growth in
5 days
Urine culture showed mixed growth
Rotavirus – negative
Ova & parasites – not performed due to improper
collection
Stool culture – negative for Salmonella, Shigella,
Campylobacter and Yersinia
Case #1
• The patient continued to have diarrhea, so the
lab received another stool for culture on day 2
of the admission
• This second stool specimen showed
nonlactose fermenting colonies on both the
hektoen enteric and Salmonella Shigella agars
Case #1
Case #1
Case #1
Case #1
• On the day the S. sonnei was identified, the
technologist checked the Campy plate
• There was growth on the Campy plate, but it
did not resemble Campylobacter
• A Gram stain and oxidase was done
• Oxidase test was positive
• Gram stain showed this:
Case #1
Case #1
• Organism was placed in the Vitek 2 for
identification and susceptibility
• Next day, an organism was identified but not
with a high confidence level
• Supervisor instructed the technologist to set
up two API 20E strips, one using the saline
ampule and the other using distilled water
Case #1
• After overnight incubation, the API 20E that
was inoculated using the saline ampule gave
an identification level of very good
• The API 20E inoculated with distilled water
gave an identification of “good likelihood, low
selectivity”
• These two API 20Es proved the halophilic
nature of the organism in question
Case #1
Case #1
• This isolate was sent to the NYC Department
of Health for confirmation
• Confirmed as Vibrio cholerae serogroup 01
Case #1
• Vibrio cholerae is the causative agent of
cholera; also known as Asiatic cholera or
epidemic cholera
• Disease probably originated in Asia,
specifically India; the Ganges River may serve
as a reservoir source for the organism
• In 1853, John Snow made the connection
between drinking contaminated water and
acquiring cholera during a London outbreak
Case #1
• Robert Koch discovered the organism in 1883
• Since its discovery, V. cholerae has caused at
least 7 pandemics
• Pandemics have stretched across Asia,
Europe, Africa and South America
• The latest outbreak of cholera is in Haiti
• Cases of cholera are not commonly reported
in the United States
Case #1
• Cases seen here are considered “imported”
cases
• Cholera is an acute diarrheal disease that is
spread mainly through contaminated water
• V. cholerae are Gram negative rods known for
their characteristic comma-shaped
morphology and motility through a single
polar flagella
V. cholerae with polar flagella
Case #1
• As a group, the possess a somatic or “O”
antigen and a flagellar or “H” antigen similar
to the Enterobacteriaceae family
• Their somatic antigen is used to differentiate
between pathogenic and nonpathogenic
strains
• Currently 139 serotypes based on the somatic
antigen have been identified
Case #1
• V. cholerae serotypes 01 and 139 are known
to cause epidemic cholera
• Serotype 01 can be further differentiated into
the El Tor and classic biotypes
• The El Tor biotype is responsible for most
cases of cholera in the world
Case #1
• V. cholerae cannot survive in the acid
environment of the stomach, so a high
infective dose of 103 to 106 organisms is
required
• Once in the small bowel, V. cholerae adheres
to the intestinal epithelial cells
• As they grow, they produce an enterotoxin
which begins a cascade of events within the
intestinal cells leading to massive diarrhea
Case #1
• Vibrio cholerae is an aquatic organism with
humans as its only known host
• Prefers salt water but can be found in
freshwater contaminated with human waste
• Has the ability to tolerate an alkaline
environment that kills most organisms
• Outbreaks have been associated with the
warm seasons and poor sanitation
Case #1
• Cholera enterotoxin binds to the intestinal
cells and stimulates these cells to
hyperproduce adenylate cyclase from ATP
• This in turn causes the hyperproduction of
cyclic AMP (cAMP)
• High levels of cAMP cause Cl, Na, K and HCO3
ions to be pumped out of the cell; water
follows the ions out
Case #1
• The bowel cannot absorb the steady and
excessive release of water and massive
diarrhea results
• Stools do not contain fecal RBCs or WBCs
because the organism does not invade and
damage the intestinal mucosa
Case #1
• The disease presents in acute cases as a
severe gastroenteritis accompanied by
vomiting followed by diarrhea
• Stool produced by patients with cholera are
described as “rice water”; they are watery and
contain numerous flecks of mucus
• Stools are also numerous and may be as may
as 10-30 per day
Case #1
• If left untreated, cholera can result in a rapid
fluid and electrolyte loss that leads to
dehydration, hypovolemic shock, metabolic
acidosis and death in a matter of hours
• An important step in the treatment of cholera
is the rapid replacement of fluid and
electrolytes lost due to diarrhea and vomiting
Case #1
• Patients who receive adequate fluid
replacement are more likely to survive even
without antibiotics
• Once fluids are replaced, antibotics are given
to shorten the length of illness and the
excretion of the organism
• Drugs of choice are tetracycline, doxycycline,
erythromycin and ciprofloxacin
Lessons Learned
• When the lab reported a pathogen, Shigella
sonnei, the physician was not impressed
• The physician had a suspicion that the patient
had Vibrio cholerae based on the country the
patient came from
• When the lab reported V. cholerae, he was
satisfied with the results
• This is a case where communication with the
lab would definitely have helped
Lessons Learned
• If the physician had told the lab what he
suspected, selective media could have been
used
• TCBS (thiosulfate citrate bile salts sucrose)
agar is the medium of choice for the isolation
of V. cholerae
• This medium differentiates the sucrose
fermenting species such as V. cholerae from
those that do not
Case #2
• 35 year old woman presented to the ED with a
chief complaint of upper gastric pain
• Patient had just returned to the US after
visiting family in Equador
• This patient was being followed in the medical
clinic for cholecystitis
• Due to her symptoms she was advised to have
surgery for the removal of her gall bladder
Case #2
• After surgery, she developed a fever and so
two sets of blood cultures were drawn and
sent to the lab; blood cultures were incubated
in the BacT/Alert
• She continued to have fever, so more blood
cultures were sent to the lab; in total the lab
received six blood culture sets
• The first sets of blood cultures became
positive at 4.9 days of incubation
Case #2
• A Gram stain was prepared from the positive
blood culture bottles
• The Gram stain showed the following:
Case #2
Case #2
• As per the lab’s protocol, the Gram stain
results were called to the nursing unit
• Blood culture bottles were subcultured to:
blood agar, MacConkey agar, CNA agar and
chocolate agar
• After overnight incubation, the following
growth was seen:
Case #2
Case #2
• The technologist working on this culture at
first thought it might be Haemophilus due to
its morphology on Gram stain
• After observing growth of the plates, she
made another Gram stain
• After performing the Gram stain she
performed three additional tests: catalase,
oxidase and urease
• All tests were positive
Case #2
• Based on the faintly staining Gram negative
coccobacilli that were catalase, oxidase and
urease positive, a presumptive identification
was made
Case #2
Case #2
• The isolate was sent to the NYC Department of
Health for identification
• The isolate was identified as Brucella
melitensis serogroup 3
• Brucella is a potential bioterror agent and
brucellosis is a nationally reportable disease
Case #2
• Brucella are intracellular bacteria that are
usually found in animals, with humans being
accidental hosts
• First isolated by Sir David Bruce on the island
of Malta
• Brucellosis is acquired through aerosol,
percutaneous and oral routes of exposure
Case #2
• Based on DNA analyses, it now seems that
there is only one species, B. melitensis, with a
number of biovars
• Brucella are, however, often still referred to by
their original “species” designations
• The 4 that are known to infect humans each
has a typical animal reservoir
Case #2
•
•
•
•
•
B. melitensis – goats and sheep
B. suis – swine
B. abortus – cattle
B. canis – dogs, especially beagles
The disease in humans is most often referred
to as brucellosis, but also is known as Malta
fever and undulant fever
Case #2
• Clinical presentation of the illness are similar
of all routes of exposure
• There are three clinical stages of brucellosis:
acute, sub-chronic and chronic
• Symptoms of acute infection are nonspecific
and include fever, malaise, headache,
anorexia, myalgia and back pain and usually
occur within eight weeks of exposure
Case #2
• This patient acquired her Brucella infection by
the oral route
• Her family in Ecuador lived in a rural area and
they regularly ate products such as milk and
cheese made from the milk of sheep
• This patient acquired the disease the “usual”
way and not due to bioterrorism
Case #2
• Brucella are disseminated via the lymphatics
and the bloodstream to many parts of the
body
• The fever may have a daily periodicity rising in
the afternoon and falling at night; this
undulatant fever is seen primarily after
infection of B. melitensis
Case #2
• One of the most interesting parts of this case
was the fact that the blood culture was even
positive during the 5 days of incubation in the
BacT/Alert
• For the isolation of Brucella, the laboratory is
usually asked to hold the blood cultures for a
longer period of time, usually 21 days
• In acute cases, 50-80% of blood cultures will
be positive
Case #2
• Brucella can also be acquired by aerosol and a
number of laboratory-acquired cases have
been reported
• Brucella should be handled under biosafety
level 3 conditions; this means the use of a
biological safety cabinet
Lessons Learned
• Always use a biological safety cabinet until
you have some indication that the organism is
one that you cannot acquire by aerosol
• This organism was examined on the open
bench and also placed in the Vitek 2 and was
vortexed during inoculum preparation
• As a result, when the organism was identified
as Brucella melitensis, several technologists
spent several weeks on antibiotics
Lessons Learned
• After this incident, all positive blood cultures
were examined first in a biological safety
cabinet during Gram stain preparation and
when the plates are initially read
Case #3
• A 42 year old man was seen in the ED of a
community hospital
• His chief complaint was fever
• He had recently returned to the US from India
where he had visited his family
• In the ED, two blood cultures were drawn and,
because he appeared quite ill, he was
admitted
Case #3
• The blood cultures were drawn at night when
the microbiology laboratory was closed
• These cultures were incubated in a small
incubator that was outside the microbiology
lab
• The next morning the blood cultures were
placed in the BacT/Alert
Case #3
• At about lunch time, these blood cultures
were flagged by the instrument as positive
• The technologist made a Gram stain of the
bottles and these organisms were seen
Case #3
Case #3
• The nursing unit was called with the Gram
stain report
• The blood culture was subcultured to blood
agar, MacConkey, CNA and chocolate agars
• After overnight incubation, the following
growth was seen
Case #3
Case #3
• As per the laboratory protocol, the organism
was placed in the Vitek for identification and
susceptibility
• The identification was excellent, but it was an
unusual isolate
• Some further testing was done; serology
Case #3
Case #3
Case #3
• Salmonella typing gave these results initially:
o Salmonella polyvalent antiserum – negative
o Salmonella Groups A, B, C1, C2,D and E –
negative
o Salmonella Vi – positive
These serological results indicate one
particular species of Salmonella
Case #3
Case #3
• To specifically type this Salmonella you need
to make a suspension of the organism in saline
and heat to 100oC for 10 minutes
• This inactivates the capsular Vi antigen and
allows you to type the organism
• Cooled suspension was again typed and this
time agglutinated in the polyvalent as well as
the Group D antisera
Case #3
• Salmonella typhi was set up on some
biochemicals as per the laboratory’s protocol
• The reaction were:
Case #3
Case #3
• Salmonella was first discovered more than 100
years ago by an American scientist named
Salmon
• Salmonella is a complex genus of
Enterobacteriaceae that contain more than
2,400 different serologic types
• These types are based on the O, H and Vi
antigens
Case #3
• O or somatic antigen is found on the surface
of the bacterial cell
• Somatic antigens allow grouping of Salmonella
to the group level
• H or flagellar antigen is, of course, found on
the flagella
• Flagellar antigens are used to speciate the
Salmonella
Case #3
• Vi or capsular antigen can sometimes mask
the O or somatic antigen as in this case
• Capsular antigens are heat labile and so are
destroyed during heating and allow the
organism to be serologically grouped
Case #3
• Salmonella typhi is the major cause of typhoid
fever
• Each year approximately 400 cases are
reported in the US, with 70% having been
acquired while traveling outside the country
• In developing countries, the disease occurs in
an estimated 12.5 million people
• S. typhi is found exclusively in humans
Case #3
• A large number of these organism must be
ingested; they are susceptible to the acidity of
the stomach
• If viable organisms reach the small intestine,
they proliferate in the Peyer’s patches
• From there the organisms travel through the
lymphatic and blood and disseminate to many
parts of the body
Case #3
• After an incubation period of about 2 weeks,
fever rises to a high plateau and may stay high
for 4-8 weeks in untreated cases
• Blood cultures are positive during the first and
second weeks after infection
• Stool cultures are positive from the second
week on
• Mortality is approximately 10-15% without
treatment
Case #3
• With treatment, mortality is less than 1%
• A carrier state is seen in 2-5% of individuals
with S. typhi
• Major reservoir in carriers is the gall bladder
because bile is a good culture medium for
these organisms
• The gall bladder dispenses bile into the small
intestine and seed the intestine with S. typhi
Case #3
• Carriers usually shed the bacteria into the
stool; this can occur intermittently
• The most famous carrier of S. typhi was
“typhoid Mary”
• All Salmonella are reportable infections to the
department of health
Lessons Learned
• S. typhi is usually seen in blood cultures first
• It may be seen in the stool but usually later on
in the disease
• Small amounts of H2S may be difficult to see in
media such as TSI
• If any organism appears biochemically to be
Salmonella typhi, correct serological typing is
essential
The Big Picture
• Communication between the microbiology
laboratory and the physician is essential
• If the physician suspects an unusual organism
that the laboratory generally does not look for,
it is even more important that the laboratory
is informed
• Communication can also enable the lab to
isolate the cause of the patient’s illness in a
more timely manner
Questions?