Case

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Case 1

• 34 year old male with AML status-post allo-BMT
  complicated by severe GVHD
• Admitted with lower fevers, GI bleed and
  diarrhea.
• Nodule noted in right lower lobe of lung and a
  bronchoscopy was performed.
• Modified acid fast stain requested.

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BAL-Modified Acid Fast Stain
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Case 1
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Identification of Fungi
lt1um Branching Filaments (actinomycetes)
Filamentous fungi
Yeast
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Actinomycetes

• Resemble fungi in that they form filaments but
  their lack of mitochondria/membrane bound nucleus
  and susceptibility to anti-bacterial agents
  define them as bacteria.
• Includes Nocardia, Nocardiopsis, Actinomadura,
  Streptomyces.
• Initial distinction made on basis of partial
  acid-fast stain followed by chemical tests.
• Nocardia are acid-fast positive.

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Modified Acid Fast
Gram Stain
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Nocardia

• Can cause pulmonary, systemic or cutaneous
  disease.
• Special request for modified acid-fast stain.
• Special request for Nocardia culture.
• Grow on Sab agar and LJ agar
• White, orange chalky colonies
• Will grow on blood, chocolate but can take over a
  week and normal sputum culture plates only held
  48 hours.
• After suspect an actinomycetes will do further
  tests to determine genus/species (e.g., lysozyme
  sensitivity, casein decomposition)

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Nocardia
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Case 2

• 23 year old woman presents with worsening left
  groin pain.
• At age 15 developed cough and fever while living
  in Arizona which cleared on its own.
• CT scan shows retroperitoneal abscess from which
  fluid was aspirated and sent for cytology and
  fungal culture.

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Case 2
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Case 2
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Case 2 Abscess Fluid
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Csae 2 Abscess Fluid
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Identification of Fungi
lt1mm Branching Filaments (actinomycetes)
Filamentous fungi
Yeast
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Filamentous Fungi
Hyaline monomorphic
Septate hyphae
Aseptate hyphae
Saprophytes -Aspergillus -Penicillium -Fusarium -S
copulariopsis -Others
Dermatophytes -Microsporum -Trichophyton -Epidermo
phyton
Hyaline Dimorphic -Histoplasma -Coccidioides -Othe
rs
Zygomycetes -Rhizopus -Mucor -Absidia -Cunninghame
lla -Others
Dematiaceous (dark mold) -Exophiala -Clodosporiu
m -Others
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Dimorphic Fungi

• Grow as filamentous molds in the environment or
  when cultured on routine mycology agar at 25-30
  degrees C
• Yeast-like in vivo or when grown on enriched
  media at 35-37 degrees C
• Includes Histoplasma, Coccidioides,
  Paracoccidioides, Blastomycoces, and Sporothrix

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Coccidioides immitis

• Endemic in Southwestern US, Central and South
  America
• Typically asymptomatic or self-limiting
  respiratory infection although can infect almost
  any tissue.
• Growth typically in 3-5 days although
  characteristic arthroconidia (barrel-shaped
  alternated with empty cells) may take 1-2 weeks
  to grow. White cottony colony.
• Appears as spherule filled with endospores in
  tissue. Can produce spherules in vitro but need
  special conditions and not typically done.
• Infection can be confirmed by exoantigen testing
  or nucleic acid probes.

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Coccidioides-fungal form
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Spherules and Endospores
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Histoplasma capsulatum
Blastomycosis dermatididis
Coccidioides immitis
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Case 3

• 78 yo female presented to her PCP in 1999 with
  cough. Labs showed eosinophilia. CXR showed
  infiltrates. AFBx3 negative.
• Eosinophilia decreased but developed
  bronchiectasis and pulmonary nodules.
• Culture obtained from open lung biopsy.

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MGIT System

• Middlebrook media
• Fluorescent compound on bottom that is quenched
  by oxygen in the tube.
• As mycobacteria consume oxygen, fluorescence can
  be detected.
• Most of MAC detected in nine days while MTB by 14
  days.
• Follow up with acid fast stain.
• Can test if fast grower by growth on MacConkey
  minus crystal violet
• If growth, do further chemical tests (e.g,
  nitrate)
• Otherwise, Gen-Probe

http//labmed.ucsf.edu/CP/SFGH/Microbiology/images
/MAI.jpeg
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Gen-Probe

• Start with Gen-probe to MTB, MA, MI
• If negative, M. kansasii, gordonae
• If unidentified, send to State Lab
• All antibiotic testing done as send-out.

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MAI

• Major clinical scenarios in HIV negative
  patients.
• Smoker with COPD, clinically like TB
• Patient with known bronchiectasis
• Elderly woman with infiltrates (Lady Windermere
  Syndrome)
• CT often reveals small nodules

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Case 3 continued

• Patient presents in 2/2003 with worsened cough
  and sputum production.
• Sputum sent for culture.

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Identification of Fungi
lt1mm Branching Filaments (actinomycetes)
Filamentous fungi
Yeast
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Filamentous Fungi
Hyaline monomorphic
Septate hyphae
Aseptate hyphae
Saprophytes -Aspergillus -Penicillium -Fusarium -S
copulariopsis -Others
Dermatophytes -Microsporum -Trichophyton -Epidermo
phyton
Hyaline Dimorphic -Histoplasma -Coccidioides -Othe
rs
Zygomycetes -Rhizopus -Mucor -Absidia -Cunninghame
lla -Others
Dematiaceous (dark mold) -Exophiala -Clodosporiu
m -Others
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Pseudallescheria boydii
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Pseudallescheri boydiiScedosporium
apiospermumGraphium

• All the same organism
• S. apiosperum and Graphium are asexual stages.
• P. boydii is the sexual stage and you see
  cleistothecia

P. boydii
Graphium
S. agiospermum
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Pseudallescheria boydii

• Most commonly a cause of mycetoma
• Can also infect other tissues (e.g., bone, lung)
  or be disseminated particularly in
  immunocompromised patients.
• Resistant to amphotericin B

Khurshid, A, et al., Chest (1999), 116, 572.
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Case 4

• 55 yo male with newly diagnosed pulmonary
  fibrosis.
• Right middle lung biopsy showed usual
  interstitial pneumonitis with active inflammatory
  component c/w possible recent infections or toxic
  insult.
• Biopsy also sent for culture.

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Filamentous Fungi
Hyaline monomorphic
Septate hyphae
Aseptate hyphae
Saprophytes -Aspergillus -Penicillium -Fusarium -S
copulariopsis -Others
Dermatophytes -Microsporum -Trichophyton -Epidermo
phyton
Hyaline Dimorphic -Histoplasma -Coccidioides -Othe
rs
Zygomycetes -Rhizopus -Mucor -Absidia -Cunninghame
lla -Others
Dematiaceous (dark mold) -Exophiala -Clodosporiu
m -Others
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Aspergillus
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Aspergillus terreus

• Cinnamon-brown colony
• Biseriate phialides, compactly columnar
• Aleuriospores found submerged in agar

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Aspergillus niger

• Black colony
• Biseriate phialides which cover entire vesicle
  and radiate

http//www.iums.org/ICPAAspnig.htm
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Aspergillus flavus

• Light green to brown colonies. Produce
  aflatoxin.
• Uniseriate and biseriate phialides that cover
  entire vesicle and point out in all directions.
• Orange growth on Aspergillus differentiation
  media.

http//www.apsnet.org/online/archive/1998/pean074.
htm
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Aspergillus fumigatus

• Velvety, dark green colonies.
• Uniseriate phialides, usually on upper 2/3 of
  vesicle.
• Also can grow at 50 degrees.

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Case 5

• Recently homeless male
• Presented to primary doctor with dystrophic
  toenails and tinea pedis not responsive to OTC
  anti-fungals.
• Toenail sent for culture.

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Filamentous Fungi
Hyaline monomorphic
Septate hyphae
Aseptate hyphae
Saprophytes -Aspergillus -Penicillium -Fusarium -S
copulariopsis -Others
Dermatophytes -Microsporum -Trichophyton -Epidermo
phyton
Hyaline Dimorphic -Histoplasma -Coccidioides -Othe
rs
Zygomycetes -Rhizopus -Mucor -Absidia -Cunninghame
lla -Others
Dematiaceous (dark mold) -Exophiala -Clodosporiu
m -Others
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Scytalidium dimidiatum

• 20 year old Pakistani female rice-field worker.
• Disease began 6 years ago.
• Does not grow on mycobiotic agar (due to
  cycloheximide.
• 50 improvement after 6 weeks of treatment with
  ketoconazole.

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Scopulariopsis
Annellide
Conidia

• Hyaline, monomorphic, saprophyte.
• Often a contaminant but can cause nail infection.
  Rarely soft tissue, bone or lungs in
  immunocompromised.

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Case 6

• 51 yo female s/p double lung transplant presents
  with productive cough
• CXR showed clear lungs
• Sent for flexible bronchoscopy with culture.

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Bordetella bronchiseptica

• Gram negative pleomorphic rods.
• Unlike B. pertussis, will grow on blood and often
  MacConkey.
• Cause of kennel cough (dogs), snuffles (rabbits),
  atrophic rhinitis in pigs.
• Also know to cause pertussis like syndrome in
  humans especially those exposed to animals and
  immunocompromised hosts.

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Bordetella

• Antibiotic susceptibility
• B. pertussis and parapertussis usually sensitive
  to erythromycin.
• B. bronchiseptica is resistant to erythromycin.

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Case 7

• 34 yo male with AIDS presents with dizziness,
  nausea and vomiting.
• Develops diarrhea while in hospital.

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Microsporidium
Trichrome stain
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Microsporidium
Legend Pt Portion of coiled polar tubuleEx
Electron dense exosporeEn Electron lucent
endosporePv Posterior vacuole
Species by EM Treatment albendazole
http//www.zoo.utoronto.ca/dgodt/Microspridia20EM
.html

http//www.dpd.cdc.gov/dpdx/HTML/ImageLibrary/Micr
osporidiosis_il.asp?bodyM-R/Microsporidiosis/body
_Microsporidiosis_il_th.htm
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E. Intestinalis vacuole
Eukaryotic cell bursting and Releasing E. hellem
spores
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Case 8

• 69 yo patient found unresponsive outside after
  housefire. Intubated in field, soot in airway.
• Brought to BWH for burn management.
• Developed fever while in SICU. CT scan showed
  free gas in peritoneum.

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Anaerobic Isolation
Growth in anerobic blood culture bottle
Plate on CNA, KV and Brucella plates
for anaerobic growth
No growth
Gram stain. Plate on blood, chocolate and
McConkey and grow aerobically.
Gram stain, plate with kanamycin, colistin and
vancomycin discs
Gram postive, Boxcar shaped rods
Growth
Look for other features suggestive Of C.
perfringens RapidANA.
Work-up for aerobic culture.
Double zone of Beta-hemolysis
Lecthinase production
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Clostridium perfringens

• Box-car shape
• Lecithinase positive on egg-media.
• Double zone beta-hemolysis
• Sepsis characterized by spherocytes with severe
  intravascular hemolysis.

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Case 9

• 18 yo Harvard student developed sore throat.
• Heterophile positive. Very severe sore throat
  treated with steroids.
• Presented one month later with SOB and pain in
  right shoulder. CXR showed RLL pneumonia with
  effusion (empyema).
• Culture of effusion grew Streptococcus.
  intermedius.

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Case 9

• 18 yo Harvard student developed sore throat.
• Heterophile positive. Very severe sore throat
  treated with steroids.
• Presented one month later with SOB and pain in
  right shoulder. CXR showed RLL pneumonia with
  effusion (empyema).
• Culture of effusion grew Streptococcus.
  intermedius.

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Strep and Staph
Streptococci in sputum sample
Staphylococci in wound sample
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Isolation of Strep and Staph
Gram cocci
Clusters, catalase
Chains, catalase -
Staphylococcus
Streptococcus, Enterococcus
Coagulase
Coagulase -
S. aureus
R
S
Novobiocin
S. saprophyticus
S. epidermidis
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Coagulase Test

• Initial test is Staphaurex
• Fibrinogen coated particles to detect bound
  coagulase.
• IgG coated particles to detect protein A
• Sensitivity 99.8
• Should do tube coagulase to detect free coagulase
  if high clinical suspicion of S. aureus
• Specificity 99.5
• False positives with S. saprophyticus
• Tube coagulase test (some strains only produce
  free coagulase)
• In plasma
• Look for clumping after 4 hours.
• To detect weak coagulase producing strains, read
  again at 18 hours. Strong producers can look
  negative at this point.

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Isolation of Strep and Staph
Streptococcus, Enterococcus
Hemolysis on sheep blood agar
Alpha
Gamma
Beta
Optochin, Bile solubility
Strepstrip
Bile esculin High salt growth PYR test
Type Also, Grp A bacitracin sensitive.
Sensitive, soluble
Viridans Strep
Enterococcus
S. pneumoniae
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Alpha-hemolytic Streptococci