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MICROBIOLOGY INFECTIOUS DISEASE
CASE STUDIES
PATHOLOGY SMALL GROUP
2012
Compiled by the Faculty
Department of Molecular Genetics and Microbiology
Modified by the Faculty
Department of Pathology and Laboratory Medicine
Duke University Medical Center
Durham, North Carolina
MENINGITIS
CASE HISTORY: An 18-year old college freshman presents to student health with a fever,
malaise, a purpuric, non-blanching rash and nuchal rigidity. A lumbar tap was performed with
the following results: cell count was 300, 100% PMN; glucose 10mg/dl; protein 100 mg/dl; and
Gram stain revealed numerous PMN’s with intracellular gram-negative diplococci.
1. What is the most likely organism in this case?
2. What type of medium would be required to isolate this organism?
CASE HISTORY: A 13 month-old child was running a temperature at home when he
suddenly had a grand mal seizure with shaking of arms and legs. The mother called 911 and the
EMT's found the child limp, unresponsive. In the emergency room he had a second seizure. He
was lethargic, neck was supple, temperature was 39.5C (103.1F). Blood cultures and lumbar
tap were performed. CSF findings were as follows: cell count was 4000, 95% PMN's; glucose
20 mg/dl; protein 125 mg/dl; Gram stain revealed numerous PMN’s and occasional
coccobacillary Gram-negative rods. Social history revealed that the family had immigrated
from another country two months prior.
1. What is the most likely organism in this case?
2. What is the significance of the social history to the organism that was isolated?
CASE HISTORY: The patient was a 34-year old HIV-positive male with a CD4 count of
80/L. The patient had a 1-week history of progressively worsening headache, photophobia,
lethargy, and fevers to 38.5°C. On the morning of his admission, he became confused,
disoriented, and ataxic, having fallen three times. On physical examination, he was lethargic
and could only answer a few questions before falling asleep. His vital signs were all within
normal limits. Chest examination and radiograph were normal. He had a head CT scan,
which was also normal. Because of his decline in mental status and history of headache and
photophobia, a lumbar puncture was done. It showed 32 WBC/L with 89% lymphocytes
and 6% monocytes, a glucose level of 22 mg/dl, and protein of 89 mg/dl. Gram stain of his
CSF revealed spherical yeasts. Serum and CSF tests for the presence of a specific antigen
were positive. The organism was recovered from cultures of his CSF and blood.
1. What is the organism most likely to be causing his illness?
2. Are his CSF cell count and chemistries consistent with infection with this organism?
3. What other organisms are frequently seen causing CNS infections in this patient
population?
4. What is the specific antigen that was found in his serum and CSF?
5. In which clinical situations can this antigen test be used to help manage patients.
6. What other organism will give a positive reaction in this test?
7. How did this patient become infected?
8. How should this patient be managed to prevent relapses with this organism in the
future?
DISCUSSION
Infections of the central nervous system constitute the real emergency situation in
clinical microbiology. Patients can die in a matter of hours of presenting to the emergency
room. The only cost effective, truly rapid, microbiological test that can effect treatment is the
Gram stain. In both cases presented here the Gram stain was positive for bacteria. In one study
from Bowman Gray School of Medicine, the Gram stain was positive in 80 to 100% of the
cases of N. meningitidis, S. pneumoniae and H. influenzae, but only positive 25% of the time
for group B streptococcal and other bacterial meningitides.
Other surrogate markers of infection are the concentration of protein, glucose, and the
type and number of cells in the CSF.
ENDOCARDITIS AND IATROGENIC INFECTIONS
CASE HISTORY: The patient is a 45-year old man who had his mitral valve replaced with a
Starr-Edwards valve one month ago. He presents with a temperature of 38.6C (101.5F),
splinter hemorrhages in his nails, and a new heart murmur. Trans-thoracic echocardiogram
revealed 2 cm vegetation on the valve. A total of 3 blood cultures were taken over a two hour
period of time and sent to the laboratory. After 10 hours the laboratory notified the physician
that 2 out of 3 of the blood cultures grew Gram-positive cocci in clusters.
1. What is the most likely organism in this case?
2. What are the pre-disposing factors which led to the patient becoming infected?
3. What is the most likely source of the organism?
3. Was the correct or incorrect number of blood cultures drawn on this patient?
4. Why did the clinician collect the cultures over a two hour time period?
CASE HISTORY: The patient was a 25 year-old international graduate student. The patient
presents with a temperature of 38C (100.4F), malaise, and generalized weakness. Significant
laboratory findings include a white count of 16,700 with 85% PMN's and negative Monospot
test. Physical exam revealed a well-nourished young male in moderate distress. Lungs were
clear and there were normal bowel sounds. A grade II/VI grade systolic murmur was heard
over the left sternal border and an echocardiogram revealed a 1 cm vegetation on the aortic
valve. Past medical history was significant for a history of rheumatic fever and extensive dental
work 2 months prior to presentation. Three blood cultures were drawn and sent to the
laboratory. The patient was admitted and begun on what was later determined to be the
appropriate antibiotic. After 24 hours all three cultures were positive for a Gram-positive
coccus in chains.
1. What is the most likely organism in this case?
2. What are the pre-disposing factors which led to the patient becoming infected?
3. What is the most likely source of the organism?
4. What was the appropriate antibiotic that was used on this patient?
CASE HISTORY: This 67-year old woman was admitted with a recurrence of her poorly
differentiated squamous cell carcinoma of the cervix. She underwent extensive gynecologic
surgery and was maintained postoperatively on broad-spectrum intravenous antibiotics. The
patient had a central venous catheter placed on the day of the surgery. Beginning 3 days
postoperatively, the patient had temperatures of 38.0°C to 38.5°C, which persisted without a
clear source. On day 8 postoperatively, she had a temperature of 39.2°C. Cultures of blood
and of the tip of the central line grew a yeast that was ovoid and reproduced by budding. The
organism was recovered on sheep blood agar medium.
1.
What is the differential diagnosis of this patient’s infecting organism?
2.
The organism was subsequently shown to form germ tubes. What is the organism?
3.
Is this organism part of the normal flora in humans?
4.
What factors in the patient’s history predisposed her to infection with this organism?
5.
What was the likely source of the organism causing this infection?
DISCUSSION
Bacteremia occurs during the course of many diseases such as pneumonia, urinary tract
and wound infections, meningitis and even some diarrheal diseases. At times, as in the two
cases in this exercise, the vascular system is the primary site of the infection.
The vascular system usually does not serve as the primary site of infection unless there
is some underlying condition. In case one, the patient had surgery with a foreign body replacing
the native valve. Foreign bodies and manipulation predispose one to infections of the vascular
system. In this case, this infection was acquired during surgery and the organism, coagulase
negative Staphylococcus, is a major cause of surgical infections when a foreign body is present.
In the second case, the damage to the vascular system was due to rheumatic fever which
damages the lining of the heart and heart valves leaving them prone to deposition of fibrin.
When organisms gain access to the blood stream, as in a dental procedure, they deposit
on the fibrin and develop into the vegetation which consists of layers of organism and fibrin.
The viridans streptococci isolated in the second case are the most common cause of bacterial
endocarditis involving native valves.
In both cases the organisms live in the vegetation and are shed intermittently into the
blood stream. At times pieces of the vegetation break off and are trapped in capillaries
throughout the body. These can lead to infections in other organs, such as the brain, where they
may form abscesses. Blockage of the capillaries also causes leakage and splinter hemorrhages.
These can occur anywhere in the body but are most easily observed in the nails and
conjunctiva.
Regardless of the source of the organisms in the blood, they are often present in low
numbers and the presence is not constant over time. The average adult who is bacteremic has
only 1 to 10 organisms/1 ml of blood. This requires that one obtains a large volume of blood,
usually 10 to 20 ml, and collects at least two to three cultures to diagnose more than 95% of
patients with bacteremia. A single blood culture only detects about 85 to 90% of bacteremic
patients. Since the number of organisms in the blood is usually low, the entire volume of
blood is cultured in a broth medium. A set of blood cultures may consist of two aerobic
media or one aerobic and one anaerobic medium with 5 to 10 ml added to each bottle of
medium. (The volume may vary with the system being used in the laboratory.) Taking of
more than one set of blood cultures over time also aids in determining the significance of an
isolate. Coagulase negative staphylococci are present on skin and single isolates frequently
indicate a “contaminated” blood culture; whereas, multiple isolates over time probably
indicate that the isolation of coagulase negative staphylococci is significant. In case one, the
multiple isolates over time, coupled with a clinical history consistent with endocarditis, make
it easy to determine the significance of the coagulase negative Staphylococcus.
When the blood culture bottles reach the laboratory they are usually incubated in
instruments that monitor bacterial growth by measuring the CO2 produced or the changes in
atmospheric pressure in the bottles. These instruments generally measure the changes every 6 to
12 minutes and are termed continuous monitoring instruments. These instruments can detect
positive bloods in an average of 18 hours compared to the 42 hours needed for detection by
manual methods or older instrumentation. In some studies, positive bottles were detected in as
little as 4 to 6 hours with continuous monitoring instruments.
When a significant change is detected in the bottle, the instrument signals it as the
positive. The technologist removes the bottle, performs a Gram stain and notifies the
clinician of the results. The bottle is also subcultured to agar plates to isolate the organism for
identification and susceptibility testing. Most laboratories incubate bottles for 5 days before
signing them out as negative.
URINARY TRACT INFECTIONS
CASE HISTORY: A 25-year-old woman presents with left flank pain, frequency and burning
on urination. Urinalysis reveals 2+ white blood cells, a positive nitrite test and 2+ bacteria. No
casts were seen. A urine culture was submitted.
1. What is the likely organism in this case?
2. What does the colony count of the organism in the urine specimen indicate?
3. What do the urinalysis findings indicate?
4. Young women have more urinary tract infections than young men. Why?
5. What Gram-negative rods are lactose fermenters?
6. Which are the most common causes of UTI?
CASE HISTORY: 75-year-old male nursing home patient had been complaining of urine
retention and the feeling of being unable to completely empty his bladder. The patient also
complained of feeling flushed and was agitated. His temperature was 39C. Physical exam
revealed an enlarged prostate.
1. What is the likely organism in this case?
2. What would the laboratory report say about the colony count of the organism in the urine
specimen?
3. As men age, the frequency of UTI's increase to that of women. Why?
4. If the organism is characterized as a Gram-positive coccus, growing in short chains, and
catalase negative, what is the most likely organism?
5. What therapeutic/resistance problems may be relevant in this case?
DISCUSSION
The urinary tract infection (UTI) is one of the most common bacterial diseases.
Patients of both sexes and all age groups are affected. Proper management of these infections
requires an understanding of the number and kinds of bacteria involved.
Most UTI’s result from ascending infection by organisms introduced through the
urethra, and less commonly via the hematogenous route. In hospitals, urinary tract infections
are the most common form of nosocomial infection. Acute infections are more common in
females than in males because of the shorter urethra and greater likelihood of its
contamination. A bacterial count of 100,000 or more organisms per ml of urine from patients
who are asymptomatic for urinary tract infection is considered to be a significant bacteriuria
and suggestive of infection. This is because urine is an excellent culture medium for most
organisms that infect the urinary tract, and because growth occurs in vivo in the urine itself,
resulting in high bacterial counts in established infections. In contrast, contamination from
the external genitalia, in the absence of infection, usually contributes less than 1,000
organisms per ml in properly collected and transported specimens.
UTI’s may vary in severity from an unsuspected infection to severe systemic diseases
involving microbial invasion of any of the tissues of the urinary tract. Infection may involve a
single site, such as the urethra (urethritis), prostate (prostatitis), bladder (cystitis), or kidney
(pyelonephritis), or more than one site, as frequently occurs.
The most common sites of UTI in women are the urethra and urinary bladder, but
infection from these sites may spread to the kidneys. Enterobacteriaceae are responsible for
nearly all nongonococcal urethrocystitis, with Escherichia coli accounting for about 90% of
these cases. Other organisms associated with urinary tract infections in the female include
Klebsiella, Enterobacter, Proteus, Pseudomonas, group B Streptococcus, Enterococcus,
Staphylococcus aureus, and Candida.
In the male, chronic bacterial prostatitis is a common disease, difficult to cure, and is
often responsible for relapsing urinary tract infections. It is usually caused by Gram-negative
bacilli. E. coli is responsible for 80% of the cases, the remainder being caused by Klebsiella,
Proteus, Pseudomonas, or Enterobacter.
Pyelonephritis is an inflammatory process involving both the pelvis and parenchyma
of the kidney, which may become chronic and lead to extensive renal destruction. The
clinical diagnosis of pyelonephritis is frequently overlooked because of the absence of
symptoms. There is often a correlation between a bacteremia and an unsuspected
pyelonephritis. An indwelling catheter often leads to the development of a bacteremia,
frequently accompanied by a Gram-negative rod septicemia. The demonstration of bacteria
by culture is the only reliable means of making a definitive diagnosis. In acute pyelonephritis,
E. coli is the etiologic agent in up to 90% of the patients. The remaining cases are caused by
Klebsiella, Enterobacter, Proteus, and enterococci.
Specimen Collection. Care in collection, storage, and transport of urine specimens for
bacteriologic examination is of utmost importance. Contaminating bacteria from the
perineum can multiply in specimens standing at room temperature and invalidate the results.
Urine specimens may be collected by the: (1) clean-voided mid-stream technique, (2)
catheterization, and occasionally (3) suprapubic aspiration. The clean-voided specimen is
recommended wherever possible because it eliminates the risk of infection.
Bacteriologic Examination. The basic procedure consists of two parts: (1) estimation
of the total number of microorganisms per ml of urine, and (2) isolation, identification and
antimicrobial susceptibility testing (where indicated) of the predominant microorganisms.
PULMONARY INFECTIONS
CASE HISTORY: A 42-year-old North Carolina man was in good health until 2 months
prior to admission, when he developed a low-grade fever, myalgias, and a nonproductive
cough. He was given oral erythromycin by his local physician. After 2 weeks of therapy, his
condition had not improved. A chest radiograph revealed “right middle lobe air space
disease,” and therapy with oral ampicillin was initiated. Over the next month, his condition
worsened. He noted daily fevers, chills, night sweats, and weight loss of several pounds. One
month prior to admission, a chest radiograph showed consolidation of the right middle lobe.
A PPD skin test was negative with positive controls, and an oral antibacterial antibiotic was
prescribed. The patient’s symptoms continued, and he was admitted to the hospital. The
patient had an unremarkable travel history and no animal exposure, was a non-smoker, and
had no HIV risk factors. He worked for the power company cutting tree limbs and trees. On
physical examination he was febrile to 38.3°C. The skin examination was notable for a
tender, raised, erythematous papule (1 sq. cm.) on the bridge of the nose. A chest radiography
and subsequent computed tomogram (CT) scan revealed a densely consolidated right middle
lobe, a 3.5-cm subcarinal mass, and a small right hilar mass. Bronchoscopy was performed.
KOH examination and acid-fast, modified acid-fast, and Gram stains were negative.
Examination of the lesion using a silver stain demonstrated large, spherical budding yeasts
with a broad base connecting the parent cell and the bud.
1. What is the differential diagnosis for this patient’s pulmonary disease?
2. Which organism is causing his illness?
3. What are its epidemiology and culture characteristics?
4. This patient’s lungs and skin were involved with this infection. Which other sites are
commonly involved?
5. Was there any component of this patient’s history that might alert a physician to think of
this organism?
6. Which organisms may be detected by a KOH examination?
7. Which organisms may be detected by an acid-fast stain?
CASE HISTORY: A 37 year old male, was admitted to Duke Hospital with an increased
WBC and a peripheral smear consistent with acute leukemia. A bone marrow biopsy found
70-80% blast forms, diagnostic of acute myelomonocytic leukemia. The patient underwent
induction chemotherapy, followed by a second bone marrow biopsy, which also revealed
blast forms. After a second round of induction chemotherapy, he became profoundly
neutropenic (<100 neutrophils/L) and developed fevers without a clear source. Broad-
spectrum antibiotic therapy was administered, but his fevers persisted. Empirical IV
amphotericin B was initiated, and a subsequent chest radiograph showed new bilateral fluffy
pulmonary infiltrates. A bronchoscopy with biopsy was performed, and the specimen
demonstrated hyaline, septate hyphae with acute-angle branching. A mold was cultured from
this biopsy.
1. What is the differential diagnosis of pulmonary infiltrates in a leukemic patient?
2. What is the most likely fungus seen on the direct examination of the bronchoscopic
specimen?
3. Why was biopsy and not lavage necessary to make this diagnosis?
4. Would blood cultures have been useful in helping to establish this diagnosis?
5. Where is this fungus found in nature?
6. What predisposed this patient to this infection?
7. What other types of infections are caused by this fungus?
DISCUSSION
Mycotic infections are generally uncommon but they are important diagnostic
considerations that appear frequently on board exams.
Mycotic pulmonary infections are caused by inhalation of infectious fungal cells, which may
be found in and cultured from various environmental sources (with the exception of
Blastomyces dermatitidis and Paracoccidioides brasiliensis, whose natural reservoirs are
unproven). Some of these mycoses are limited to geographical areas of endemicity (e.g.,
coccidioidomycosis and blastomycosis) while others have a worldwide distribution (e.g.,
cryptococcosis). Therefore, a history of travel or exposure to potential fomites from an
endemic area is important. Since these mycoses can mimic tuberculosis, cancer, or other
chronic diseases, proper management of the patient depends on an early definitive diagnosis
from the laboratory.
A.
Endemic Mycoses
Blastomycosis is a primary pulmonary infection which may be limited to the lungs but
usually disseminates to the subcutaneous tissues, skin, bones and other organs. Only rare
cases occur outside the eastern half of North America. The etiologic agent, Blastomyces
dermatitidis, is a thermally dimorphic fungus.
For direct microscopic examination, clinical material, such as sputum, lung or skin tissue, is
mixed with l0 or 20% KOH on a glass slide; a coverglass is added and the preparation is
heated gently (see Appendix). In KOH preparations, calcofluor white, or hematoxylin-eosin
stained tissue sections, B. dermatitidis appears as a single-budding, spherical, yeast cell 5-l5
µm in diameter with a thick refractile cell wall. The bud and the parent cell are typically
connected by a broad base of attachment.
All clinical materials are cultured on Inhibitory Mold Agar or Sabouraud’s glucose agar at
30°C and on brain heart infusion (BHI) agar containing 5% sheep blood at 37°C. When
culturing nonsterile specimens, antibiotics should be added to the medium to inhibit bacteria.
On Sabouraud's agar at 30°C, a cottony, at first white, then buff to brown mold develops
within three weeks. The colony is composed of branching hyphae that bear spherical to
pyriform conidia on short conidiophores. On BHI agar at 37°C, a wrinkled, waxy and heaped
colony develops, which is composed of yeast cells resembling those seen in lesions.
Histoplasmosis is an acute or chronic, usually benign and self-limited infection of the lungs
or any tissues of the reticuloendothelial system. Residual calcifications may remain after
active lesions have resolved. Reactivation infection is not uncommon. Although
histoplasmosis occurs worldwide, geographic regions of high endemicity exist (e.g., OhioMississippi River Valleys), and the mold phase of this dimorphic pathogen, Histoplasma
capsulatum, can be found in specimens of soil mixed with aged guano in these areas.
Direct examination of unstained, clinical specimens is unproductive, but smears of sputum,
bronchial washings, lung tissue, bone marrow aspirates and similar specimens can be stained
with calcofluor white, Giemsa or Wright stain and examined. The budding yeast cells of H.
capsulatum are ellipsoidal and tiny (ca. 2 x 5 µm); they are found within macrophages in
reticuloendothelial granulomas. Despite the name, H. capsulatum does not possess a capsule.
Clinical specimens are cultured as described for blastomycosis. H. capsulatum grows slowly,
producing a cottony, initially brownish mold colony. Some isolates require up to 8 weeks or
longer. The LPCB preparation should reveal large, spherical, echinulate macroconidia and
smaller microconidia. The identification of H. capsulatum is confirmed by demonstrating
conversion to the yeast form, which is accomplished by culturing the isolate on a rich
medium (e.g., BHI with blood) at 37°C. Under these conditions, a moist, whitish colony of
small, budding yeast cells similar to those found in tissue will develop.
Coccidioidomycosis may be an acute, but benign and self-limited respiratory infection, or a
progressive, chronic, and disseminated disease. Coccidioidomycosis is endemic in the semiarid southwestern United States, and follows inhalation of the arthroconidia of Coccidioides
immitis (or C. posadasii), which are produced in desert soil by the mold form of this
dimorphic fungus. Clinical specimens, such as sputum, gastric contents, lung tissue, spinal
fluid, pus from subcutaneous abscesses, and exudate from cutaneous lesions, are placed on
glass slides and covered with coverglasses. The material may be cleared by the addition of a
drop of KOH. In direct preparations or stained tissue, C. immitis appears as a non-budding,
spherical, (20-60 µm in diameter) thick- walled spherule, which may be filled with numerous
small endospores (2-5 µm in diameter). Small, immature spherules which lack the
endospores cannot be identified as the tissue form of C. immitis.
All clinical materials should be cultured as above. C. immitis grows quickly, often producing
an abundant cottony, aerial mycelium that is white at first but becomes tan to brown in older
cultures. As a culture becomes old and dry, the aerial mycelium fragments to give the
cultures a powdery appearance. In LPCB preparations, the hyphae are seen to develop into
chains of thick- walled rectangular arthroconidia, which are characteristically deeply stained
and separated by unstained degenerating cells. However, cultures which show this type of
microscopic morphology should be confirmed by the production of spherules in mice after
intraperitoneal injection of a saline suspension of the culture. Alternatively, demonstration of
a specific exoantigen in an aqueous extract of the culture will confirm the identification.
Production of spherules in vitro is difficult.
Every precaution should be observed when handling a culture of C. immitis, as the
arthroconidia are readily aerosolized and highly infectious. Transfers and preparations for
microscopic examination should be made in a biosafety cabinet to protect the workers and
environment.
B.
Opportunistic Mycoses
Opportunistic mycoses are very common in immunocompromised patients.
In opportunistic mycoses, both the underlying disease and its management may compromise a
patient's antifungal defense mechanisms and predispose to invasive fungal infection. The
most frequent opportunistic mycoses are systemic candidiasis, cryptococcosis, aspergillosis
and mucormycosis.
Cryptococcosis is a subacute or chronic infection which follows inhalation of the
encapsulated yeast, Cryptococcus neoformans. From the lungs, C. neoformans may spread
hematogenously to any part of the body, but preferentially to the central nervous system,
causing cryptococcal meningitis. Cryptococcosis has a worldwide distribution.
For laboratory identification, fresh preparations of sputum, cutaneous lesions, or centrifuged
spinal fluid should be examined by direct microscopic observation for the presence of
spherical, budding, encapsulated yeast cells. Specimens can be examined in water, 5 or l0%
KOH, calcofluor white, or India ink. (see Appendix).
All clinical materials should be cultured at 37°C. Nonsterile specimens are inoculated on
media containing antibacterial antibiotic(s). When pure cultures are obtained, Christensen's
urea medium and a medium containing an appropriate diphenolic substrate, such as caffeic
acid, are used to complete the identification. C. neoformans develops a mucoid, white to tan
colony on most media at room temperature or at 37°C. The spherical, thick-walled budding
cells, 5-l0 µm in diameter, are surrounded by a wide gelatinous capsule that is best seen in
India ink preparations. Production of urease is indicated by an alkaline change (red) in the pH
indicator of the urea medium. Phenol oxidase production results in the development of dark
brown yeast cells. The production of urease distinguishes all species of Cryptococcus from
Candida and most other yeasts. Rare isolates of other species of Cryptococcus also grow at
37°C but only C. neoformans produces phenol oxidase (laccase).
Candidiasis may be caused by any of several species of Candida that are members of the
normal flora of the skin, mucous membranes, and gastrointestinal tract. The natural history
and risk factors associated with each of the three forms of candidiasis (superficial, systemic,
and chronic mucocutaneous candidiasis) are different. Although Candida albicans is the
most frequent pathogen, most cases of candidiasis are caused by other species, all of which
may also cause transient or fatal infections in debilitated patients. Indeed, C. tropicalis may
be more virulent in patients with leukemia. Other pathogenic species include C. parapsilosis,
C. tropicalis, C. glabrata, and C. guilliermondii.
Direct microscopic examination of sputum, transtracheal aspirates, or bronchial washings is
best performed on fresh smears stained by Gram's method. Scrapings from the mucosa can be
examined in a drop of water, calcofluor white or KOH on a glass slide covered with a
coverglass. Skin and nail scrapings are examined in a drop of KOH. In sputum, skin,
secretions, or mucous patches of the mouth or vagina, Candida are recognized as small,
spherical to oval, budding yeast cells. Pseudohyphae, true hyphae, or both may also be
observed. Candida that are present as normal flora will appear as budding yeasts. The hyphal
forms are associated with active infection and invasion of the epidermis or mucosa.
All clinical materials should be cultured at 30°C on agar medium containing antibiotics.
Typical yeast colonies develop within 48 hours at 30°C or at 37°C; these cultures have a
distinct yeasty odor, and are white to cream-colored, soft, and easily picked up with a loop.
C. albicans differs from other Candida by producing germ tubes upon incubation, in serum at
37°C for 2 hours. C. albicans also produces pseudohyphae with characteristic
chlamydospores if streaked through a corn meal agar plate. After l to 2 days incubation, this
growth can be examined on the petri dishes with the low power objective of the microscope.
Routine isolates of Candida can be speciated by their patterns of fermentation of certain
sugars and assimilation of various carbon sources.
Aspergillosis is caused by Aspergillus fumigatus and rarely, A. flavus, A. terreus, and others.
Aspergillosis is an acute or chronic infection of the sinuses, bronchi, lungs, and other parts of
the body. In debilitated patients, invasive disease is a complication of leukemia, transplantation, treatment with steroids or cytotoxic drugs, and other compromising conditions.
Regardless of the species, Aspergillus appears in the sputum, tissue, or blood vessels as
hyaline, branching septate hyphae. Isolation by culture is necessary to confirm the genus and
species. Clinical materials are cultured on Inhibitory Mold Agar with antibiotics. A.
fumigatus grows quickly to produce a white, cottony colony, which becomes velvety and
finally greenish as conidia are produced. The typical Aspergillus conidiophore is long,
nonseptate, and swollen at the tip to form a vesicle, upon which phialides produce long
chains of small spherical to ellipsoidal conidia.
Mucormycosis (zygomycosis) may be caused by several species of fungi in the Order
Mucorales (Rhizopus, Mucor, Absidia, and others). Mucormycosis involves the nasoorbital
area, the brain, lungs, and subcutaneous tissues. Hyphae invade the walls and lumen of blood
vessels resulting in vascular thrombosis, infarction, and tissue necrosis. Ketoacidosis,
leukemia, lymphoma, and other conditions predispose to rhinocerebral mucormycosis. Burn
patients are at risk for cutaneous mucormycosis. All clinical materials, such as scrapings or
washings from maxillary sinuses, sputum, ocular exudate, necrotic tissues from skin or
elsewhere, should be examined with KOH or calcofluor white for the presence of wide,
branching, irregular, sparsely septate hyphae, which are characteristic of zygomycetous
fungi.
All specimens should be cultured at 30°C on agar with antibiotics. All of the agents, such as
Rhizopus oryzae, grow quickly and fill the test tube with a grayish, cobwebish colony that
develops black dots throughout the loose mycelium. These dark areas are sporangia that are
formed on the ends of sporangiophores, which arise from substrate hyphae opposite rhizoids.
The development of rhizoids should place the isolated fungus in the genus Rhizopus, which is
responsible for most infections. Zygomycetes, as with Aspergillus species, are identified on
the basis of morphological features, temperature restrictions, and selected physiological tests.
INFECTIONS BY ANAEROBES
CASE HISTORY: A 47-year old patient with a history of smoking, alcohol and substance
abuse was found passed out in a park, lying in a pool of his vomitus. He spent the next three
days in a detox unit in the local jail. Two weeks later he presents to the ED with a productive
cough, fever, shortness of breath and generalized malaise. Chest X-rays revealed lower lobe
pneumonia with a cavity. Patient had a foul smelling breath and extremely bad oral hygiene. A
double lumen bronchoscopy was performed and the bronchial brush was submitted for culture.
After forty-eight hours the anaerobic cultures grew a long thin Gram-negative rod and Grampositive cocci of several morphologies.
1. Were the specimens submitted to the laboratory acceptable for culturing of anaerobes?
2. How should these specimens be transported to the laboratory?
3. What are the underlying conditions that predispose this patient to anaerobic infection?
4. What is the most likely organism?
CASE HISTORY: 37-year old paraplegic was found by the visiting nurse sitting in a pool of
his own feces. He had been in this condition for several days since he was unable to move his
wheelchair to the bathroom and his in-home caregiver had to leave for a family emergency. The
visiting nurse noted that the flank from the buttock to the abdomen was discolored and had a
large area of crepitance. He was taken to the emergency room where he was admitted for
immediate surgery and debridement. Gram stain of the tissue revealed large Gram-positive rods
with no spores, no underlying tissue structure and no white cells. After forty-eight hours the
anaerobic cultures grew large, non-spore forming Gram-positive rods,that exhibited a double
zone of hemolysis on the anaerobic blood agar plate.
1. Were the specimens submitted to the laboratory acceptable for culturing of anaerobes?
2. What are the underlying conditions that predispose this patient to anaerobic infection?
3. What is the most likely organism?
DISCUSSION
Anaerobic infections are generally caused by organisms that are part of the patient’s
own flora. When the integrity of the mucus membranes or skin is compromised these
organisms can invade normally sterile tissue. If there is also some damage which impedes the
blood flow to the area one has ideal conditions in which the anaerobes can grow and develop
into an anaerobic infection over the next several days or weeks. In patient one it is highly
likely that the patient aspirated vomitus and saliva and that there was tissue destruction from
stomach acid and the organisms from the oral pharynx infected the respiratory tract. The bad
dentition also indicates a high level of anaerobic organism in this patient's oral cavity. In
patient two fecal contents probably enter the tissues that were irritated or through abrasions
or cuts in the thigh and buttocks. The foul smelling breath of patient one and the decrepitance
in patient two are clinical clues of anaerobic infection which require special procedures for
sample collection.
Specimen collection for anaerobic infections requires special procedures to by-pass the
normal flora and the use of transport devices which preserve the anaerobic environment. In
case one the normal flora was by-passed by using double lumen bronchoscope. This device has
on outer sheath that protects the inner bronchoscope and bronchial brush. It also has a glycerol
plug which further prevents organism from entering the lumen of the bronchoscope. When the
scope is maneuvered into place the inner scope is pushed into the sampling area and the
glycerol plug is pushed out of the way. The brush is then used to collect the sample. When
exiting the entire process is reversed to protect the brush as it exits the oral pharynx. The brush
is then clipped off and placed into an anaerobic transport device for delivery to the laboratory.
In patient two the tissue was obtained under surgical conditions where the external skin was
disinfected with a surgical prep and the deep tissue was collected with sterile instruments. After
placing in appropriate anaerobic transport device the tissue was sent to the laboratory.
The initial Gram stain of the specimen is key to helping make a microbiological
diagnosis. In patient two the Gram stain revealed large, Gram-positive rods with no spores -this is typical for Clostridium perfringens. The Gram stain also shows no tissue and no white
cells typical of gas gangrene or clostridial myonecrosis.
In patient one we had two organisms isolated, one with the morphology of
Fusobacterium nucleatum, a organism commonly found in the oral cavity. The Gram-positive
cocci are probably several species of Peptostreptococcus, which are common anaerobic isolates
from skin and oral cavity. This case also illustrates that there are frequently more than one
organism associated with anaerobic infections. In most similar clinical situations there would
be several more anaerobic organisms and several aerobic organisms isolated.
Regardless of the organisms isolated, therapy of anaerobic infections requires drainage
of the infected site and antibiotics. In patient one, drainage will be accomplished with changes
in posture and induction of coughing. In patient two, surgical debridement of all the necrotic
tissue is necessary.
ACTINOMYCETES
Actinomycetes are generally uncommon but they are important diagnostic
considerations that appear frequently on board exams.
CASE HISTORY: A 24-year old woman underwent a cadaveric renal transplant in
September, which was complicated by rejection and treated with antithymocyte globulin.
After discharge she was treated with prednisone 40 mg every morning and 15 mg every
evening and azathioprine 100 mg daily. She was admitted in January with fever, chills, and
cough. Chest X-ray revealed multiple small abscesses within a left upper lobe infiltrate.
Laboratory evaluation revealed a WBC of 11,560 with 88% neutrophils and a serum
creatinine of 0.9 mg/dl. Sputum examination revealed many Gram-positive branching rods,
and the specimen later grew an actinomycete. No cutaneous involvement was found. She was
treated with trimethoprim/sulfamethoxazole (TMP/SMX) at 320/1600 mg every 12 hours and
doxycycline 100 mg every 6 hours. After six months, the doxycycline was discontinued and
the TMP/SMX was decreased to 160/800 mg daily. This was continued until she returned to
hemodialysis because of transplant rejection. She remains without evidence of recurrent
infection.
1.
What conditions put this patient at risk for opportunistic infection?
2.
Name at least two genera of bacteria likely on the basis of the Gram stain.
3.
Speculate about the origin of the organism causing this infection?
4.
How would the species be determined?
5.
Was the therapy appropriate?
DISCUSSION
The term actinomycetes may refer to any of several species of bacillary, Grampositive bacteria that are able, in varying degrees, to form branching filaments. In
complexity, they range from the simple corynebacteria and mycobacteria to the filamentous
nocardioforms and streptomycetes. Species of the following genera contain medically
important actinomycetes: Actinomyces, Nocardia, Actinomadura, and Streptomyces.
These genera are Gram-positive rods that tend to grow as filaments with an average
diameter of about l µm. They include aerobic and facultative species, as well as obligate
anaerobes. The actinomycetes grow on routine laboratory media but more slowly than most
other bacteria; they have a doubling time of two to three hours.
Actinomyces species grow typically as diphtheroids, and filamentous growth is limited both
in vitro and in tissue. When filaments are produced, they readily fragment into individual
cells and short chains. They are neither sporeformers nor acid-fast; many species are
facultative anaerobes.
Nocardia species are obligate aerobes and are weakly acid-fast. Although the degree of
filamentation may be limited, most species develop an extensive network of branching
filaments. Substrate filaments firmly anchor colonies to agar media. Aerial filaments extend
above the substrate and subsequently fragment into coccobacillary forms. Fragmentation of
the aerial or substrate filaments usually occurs within a few days after their formation.
Species of Actinomadura resemble Nocardia but the former are not acid fast and the
filaments do not undergo fragmentation. The streptomycetes develop extensively branched
substrate and aerial filaments that do not fragment readily. They are aerobic, spore-forming,
and not acid-fast. This family contains several hundred species, many of which are important
antibiotic producers.
Three clinically different diseases are caused by these actinomycetes: actinomycosis,
nocardiosis and actinomycetoma.
Actinomycosis Actinomycosis is a chronic suppurative and granulomatous disease with
extensive abscess formation, which leads to multiple interlocking draining sinuses
throughout the infected tissues. The major clinical types of actinomycosis – cervicofacial,
thoracic and abdominal – are defined by the site of involvement. The most common cause of
actinomycosis is Actinomyces israelii. Less frequent agents are A. naeslundii, A. meyeri,
Propionibacterium propionicus and, rarely, several other species of Actinomyces. These and
similar bacteria are part of the normal mucosal flora of the gastrointestinal tract. Regardless
of the etiology or location of the infection, the pathology consists of tissue abscesses or
sinuses, which characteristically contain microcolonies of the bacteria embedded in tissue
debris, termed granules. Actinomycotic granules are also called sulfur granules because of
their yellowish color. The granules are reniform or lobulated and may reach 1 mm in size.
The bacterial filaments are enlarged at the periphery of the granule to give the appearance of
radial "clubs."
Pus, exudate, infected tissue or granules may be examined microscopically and cultured to
establish a diagnosis of actinomycosis. Actinomycotic granules have peripheral clubs and
contain Gram-positive diphtheroids and short filaments. Specimens should be cultured on
brain heart infusion (BHI) agar plates and incubated at 37°C in an anaerobic environment, or
preferentially, under elevated CO2. Tubes of thioglycollate broth are also inoculated and
incubated at 37°C. With 48 hours of incubation of the BHI plates, A. israelii produces "spider
colonies” as filaments form and spread radially. After a week, colonies become heaped,
whitish, and "molar tooth" in appearance. A. israelii, unlike similar diphtheroid bacilli, is
catalase negative and produces compact, nondiffuse colonies in thioglycollate broth.
Nocardiosis Nocardiosis is a primary or opportunistic pulmonary infection that follows
inhalation of the etiologic agent. The infection may metastasize to the subcutaneous tissues
and other organs of the body, especially the brain and meninges. Most cases of nocardiosis
are caused by Nocardia asteroides, followed by N. brasiliensis, N. otitidiscaviarum and other
species of Nocardia. These and other species of Nocardia reside in soil and water worldwide.
N. asteroides does not produce granules in tissue, but rather Gram-positive or partially acidfast filaments are found in exudate and tissues. Sputum, lung tissue, spinal fluid, pus from
subcutaneous abscesses or exudate from draining sinuses are stained by Gram’s method or by
an acid-fast technique employing l% H2SO4 for destaining. Gram-stained smears present
long, branching filaments that tend to stain unevenly and appear granular or beaded;
coccobacillary and diphtheroid forms are also common. Acid-fast stained smears reveal long,
branching filaments; both acid-fast and non-acid-fast stained filaments may be seen. Short,
slightly curved bacillary forms may be mistaken for mycobacteria in acid-fast stained smears
of sputum.
Clinical specimens should be cultured on routine media, such as BHI with or without blood,
at 37°C. N. asteroides develops a glabrous, irregularly folded or granular colony, which may
be yellow to deep orange in color. Occasional isolates develop a white chalky surface. Gramstained smears from these cultures show Gram-positive branching filaments, bacillary, and
coccoid forms. Some isolates are non-acid-fast; others require growth on milk or egg medium
for 2-3 weeks before developing acid-fastness. N. asteroides does not hydrolyze gelatin,
casein, tyrosine or hypoxanthine, whereas N. brasiliensis is positive in these tests. N.
otitidiscaviarum, unlike N. brasiliensis or N. asteroides, will utilize xanthine.
Actinomycetoma Actinomycetoma is a local, subcutaneous infection, usually involving the
extremities. The infection begins by traumatic introduction of any one of several
actinomycetes that reside in soil. All agents produce the same clinical disease.
Actinomycetoma is defined as tissue edema and the formation of abscesses with
interconnecting, often draining, sinuses that contain sulfur granules. Actinomycetoma cannot
be distinguished from (eu)mycetoma (maduromycosis; Madura foot), which may be caused
by various fungi; however, the appearance of the granule in the tissue and culture of the
agent permit a definitive diagnosis. Granules in the tissue or exudate from draining sinuses
will be yellowish, red or black. Actinomycetoma with white to yellow granules may be
caused by Actinomadura madurae, Streptomyces somaliensis, N. brasiliensis, N.
otitidiscaviarum, N. asteroides, or A. israelii. Actinomadura pelletieri produces a red
granule, and Streptomyces paraguayensis, a black granule. Occasionally the granules will
have clubs at the periphery and will be indistinguishable from granules formed with
actinomycosis. The observation of bacterial filaments (ca. l µm wide) distinguishes these
granules from those of eumycetoma. Tissue, exudate, pus, or granules should be cultured at
room temperature. Only A. israelii requires a rich medium and anaerobic environment.
Antibiotics should not be added to the medium. These actinomycetes are identified on the
basis of morphology, physiological tests and cell wall composition.
GLOSSARY OF MYCOLOGICAL TERMS
Conidia Asexual reproductive structures (spores) produced either from the transformation of
a vegetative yeast or hyphal cell or from a specialized conidiogenous cell, which may be
simple to complex and elaborate. Conidia may be formed on specialized hyphae, termed
conidiophores. Microconidia are small, and macroconidia are large or multicellular.
Arthroconidia (arthrospores) Conidia that result from the fragmentation of hyphal
cells (e.g., Coccidioides immitis)
Blastoconidia (blastospores) Conidial formation through a budding process (e.g.,
yeasts; Cladosporium)
Dematiaceous Fungi whose cell walls contain melanin and possess a brown to black
pigment.
Dimorphic Fungi that have two growth forms, such as a mold and yeast, which develop
under different growth conditions (e.g., Blastomyces dermatitidis forms hyphae in vitro and
yeasts in tissue).
Hyphae Tubular, branching filaments (2-10 m in width) of fungal cells, the mold form of
growth. Most hyphal cells are separated by porous cross-walls or septa, but the
zygomycetous hyphae are characteristically sparsely septate. Vegetative or substrate hyphae
anchor the colony and absorb nutrients. Aerial hyphae project above the colony and bear the
reproductive structures.
Mold Hyphal or mycelial colony or form of growth.
Mycelium Mass or mat of hyphae, mold colony.
Pseudohyphae Elongated chains of buds.
Septum Hyphal cross-wall, typically perforated.
Sporangiospores Asexual structures characteristic of zygomycetes; they are mitotic spores
produced with an enclosed sporangium, often supported by on sporangiophore.
Spore A specialized structure with enhanced survival value, such as resistance to adverse
conditions or features that promote dispersion. Spores may result from asexual (e.g., conidia,
sporangiospores) or sexual (see below) reproduction. During sexual reproduction, haploid
cells of compatible strains mate through a process of plasmogamy, karyogamy, and meiosis.
Yeasts Unicellular, spherical to ellipsoidal (3-15 m) fungal cells that reproduce by budding.