Steven D. Dallas, PhD, D(ABMM), MT(ASCP)SM

Associate Professor

Departments of Clinical Laboratory Sciences and Pathology University of Texas Health Science Center San Antonio

Microbiology Section Chief

University Hospital, San Antonio

dallass@uthscsa.edu 1

Gram variable or Gram very able?

Selected Cases

Objectives

• Describe the clinical utility of the Gram stain on specimens

from various body sites and blood cultures

• Correlate Gram stain results to expected culture growth

• Discuss specific cases where Gram stains made an

immediate impact on patient care

• Review collection and transport improvements

• Discuss/critique automated Gram stain instruments.

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Fine print disclosures: I have not taken money, pizza, pens, or note pads from any vendors in the last three years.

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Specimen

Gram stain,

30 minutes

Culture,

24 hours

Antibiotics susceptibility

8 to 24 more hours

Doug

Detect, identify, eliminate. A progression of

actionable information leading to treatment and cure!

Rapid flu test, 20 minutes

Real time PCR for some

Organisms, one hour

Bug

Drug You are here

Three points

• The gram stain may detect organisms for which it

was not originally intended.

• The Gram stain can leave us in limbo: Gram-variable

coccobacilli? Many PMNs, no organisms seen?

• Some dogs will chase anything that moves.

– Some doctors will treat anything we see or grow.

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Case 1 history • 16 yr old female with pre-B ALL (10/13)

• Altered mental status, headache, respiratory distress

• Disseminated fungal infection (another hospital)

• Head CT : hydrocephalus and sub-arachnoid hemorrhage

• Patient deteriorated, intubated and placement of external

ventricular drain

• Imaging : abnormality in the left vertebral artery concerning

for a mycotic aneurysm

• Ventricular fluid (CSF) sent for culture.

Case originally presented by Komal Arora, MD,

UTHSCSA pathology resident

San Antonio Society for Pathology meeting.

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Case 1: CSF Gram stain

Cytocentrifuged CSF 1000x

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Gram Stain

Cytocentrifuged CSF 1000x

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What do you say in your Gram stain report?

Cytocentrifuged CSF 1000x

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Case 1 clinical course

• Acute drop in blood pressure and H/H, anuria and abdominal

distension with acute intra-abdominal hemorrhage and

compartment syndrome

• Hemoperitoneum/splenic aneurysm

• Splenectomy, distal pancreatectomy, excision of mycotic

splenic aneurysm.

• Pathology report:

– Consistent with mycotic aneurysm of splenic vein branch associated

with a thrombus

– GMS stain showed medium sized fragmented non septate irregular

hyphae (Zygomyces?)

– Correlation with culture recommended.

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Case 1 GMS stain

Gram stains of different fungi

Candida

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Gram stain (yeasts)

Cryptococcus

(CSF)

Histoplasma

(blood culture) 12

Aspergillus from cornea scraping

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Mucorales, MVA leg wound

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Back to the case

• CSF Gram stain detected the organism

• Yeast in CSF is almost always Cryptococcus

• Did not look like Cryptococcus

• Cryptococcal antigen test is negative.

The point is: you can see fungi in a Gram stain if you take your time!

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If you had to guess?

a) Aspergillus

b) Fusarium

c) Malassezia

d) Exserohilum

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And the answer is:

Fusarium spp.

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Fusarium

• Fusarium species are important plant pathogens

• Fusarium species cause:

– Superficial (such as keratitis and onychomycosis)

– Locally invasive or disseminated infections

(severely immunocompromised patients)

• Widely distributed in soil, plant debris

• Portal of entry: airways, followed by the skin at site

of tissue breakdown and mucosal membranes.

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Disseminated Fusarium infection

• Two characteristics suggest the diagnosis of disseminated

fusariosis in the severely immunocompromised host:

– skin lesions (either cellulitis or metastatic lesions)

– positive blood cultures for mold.

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Lab diagnosis

• Blood cultures are frequently positive in fusariosis

• Adventitious sporulation facilitates dissemination and growth in the blood

• Cottony or wooly colonies with pink or violet surface and reverse is pigmented.

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Case 1 summary • Rare to see yeast/fungus other than Cryptococcus in CSF unless

there is a surgery, trauma, or other pre-disposing factor

• Choice of antifungal could be very different

– yeast in CSF or blood: fluconazole

– true fungi: amphotericin and/or voriconazole

• Can resemble yeast due to adventitious sporulation

• Fusarium generally does not make “death banana” morphology in clinical specimens.

• Hyphal and yeast-like structures in the same specimen is

highly suggestive of fusariosis

• Importance of communication/collaboration with anatomic

pathology.

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Case 2 hints

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Case originally presented at UTHSCSA clinical pathology conference

by August Moritz, DO, UTHSCSA pathology resident

Case 2

• 17 year old Caucasian female

• “Seizures” at 3 am and 10 am

• Felt dizzy and hot, fell, and hit head on bathtub

• URI symptoms 2x/day (cough, runny nose,

fever- 102.6 in ER). It is late November.

• Jaundice “always yellow, never really noticed

it” but sclera more yellow than usual.

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Case 2 PMH

• Seizures diagnosed 3y/ago, controlled with

Keppra 60mg BID (family hx of epilepsy on

mothers side)

• Suicide attempt with Tylenol (age 14), denies

currently

• Hx self mutilation

• Hx of EtOH, Tobacco, THC (age 14).

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Case 2 Imaging

• Abdominal: splenomegaly

• Brain MRI: unremarkable except for right

sided posterior auricular hematoma (head

trauma)

• Chest X-ray: unremarkable.

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Case 2: peripheral blood

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Case 2: Let’s draw blood cultures!

• One of first two grew a “gram negative rod” af

• So, they drew two more blood cultures

• Second two were no growth.

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Case 2: the organism

• Grew on BA, CHOC, MAC, mucoid colonies

• Very unusual Gram stain

• Catalase positive, oxidase positive

• Presumptive ID can be based on the above

characteristics.

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What is the organism?

a) Bulleidia extructa

b) Paracoccus yeei

c) Sarcina ventriculi

d) Wohlfahrtiimonas chitiniclastica

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Paracoccus yeei

First speciated in 2003

• Gram negative coccobacilli, cocci, or diplococci

• Obligate aerobes

• Often with “O”/vacuolated morphology

• Lactose non-fermenting gram negative

• Oxidase positive

• Weakly catalase positive

• Found in soil, brine, environment.

Burd, E., and Sharp, S. (2012) Photo Quiz; Paracoccus yeei. Journal of Clinical Microbiology.

0095-1137 p. 543. 30

Paracoccus yeei

• First infection diagnosed 2004

– Sepsis

– Bullous skin disease

– Peritonitis

– Keratitis

– Arthritis

– Endocarditis

Very rare reports.

(only 22 papers on pubmed search of “Paracoccus yeei”)

Funke, G,., Frodl, R., an Sommer, H. (2004) First comprehensively documented case of

Paracoccus yeei infection in a human. Journal of Clinical Microbiology. 42(7) 3366-3368.

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Paracoccus yeei

• No CLSI susceptibility interpretations

• Most infections appear responsive to

fluoroquinolones

• Most MICs appear to show susceptibility to B-

lactams, carbapenems, and aminopenicillins.

• Route of infection often difficult to determine.

Wallet, F., et al. (2010) Paracoccus yeei: a new unusual opportunistic bacterium in

ambulatory peritoneal dialysis. International Journal of Infectious Disease. 14,

e173-174.

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Case 2: source of symptoms?

• Filmarray viral respiratory PCR ordered two days

after first blood cultures.

• Are symptoms likely caused by one isolated positive

blood culture? Remember, its late November!

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Significance?

• Treat for P. yeei infection?

• Perform susceptibilities?

• Does the influenza infection or P. yeei

bacteremia explain the hematologic/smear

findings?

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Case 2: clinical decision

• Assessment and plan: Patient has influenza A which could account for recent fatigue and can cause modest drop in counts.

• Work-up for anemia including electrophoresis for rare hemoglobinopathy and also will need work-up for membrane defect.

• Cannot rule out infiltrative process such as leukemia, fibrosis or other invasive process which could cause teardrop cells on peripheral smear. However, would watch over next few days to see if counts trend upwards as flu symptoms resolve.

• Patient told to stop taking Keppra

• Patient placed on tamiflu.

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Case 2: RBC explanation?

Alkaline hemoglobin electrophoresis reveals increased hemoglobin F (5.0%)

and hemoglobin A2 (measured at 5.4% by chromatography). Iron studies are

not suggestive of iron deficiency (ferritin = 443 ng/dL; iron transferrin

saturation = 34.0%). These results are consistent with beta thalassemia

minor; the elevated levels of hemoglobin F suggest that that hereditary

persistence of fetal hemoglobin is present.

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Case 2: follow-up

• Same peripheral smear findings with

microcytic anemia with dacrocytes,

spherocytes, schistocytes, tear drops,

macrocytes

• No blasts

• Patient feels great

– “just completed an Insanity workout without

issue”

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• Paracoccus is rarely isolated but has been

documented to cause infections

• The source of Paracoccus is unclear, possibly

the soil

• Paracoccus is usually susceptible to

fluoroquinolones, B-lactams, and carbapenems.

Case 2 summary

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Case 3: Unknown male, unknown bug

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People look different in liquid compared to dry land. So do microbes. Growing in liquid they are described as planktonic.

On an agar surface, the growth is similar to a biofilm.

Five humans growing in water Same five humans growing on dry land

Case 3

• Unknown male

• Blood cultures X 2

• One set grows GPC in clusters (left arm)

• Other set grows GPC in clusters and a weird

bug (right AC line)

• Final of first set: S. aureus and CNS

• Second set: S. aureus and weird bug

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Case 3: how to report?

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Case 3: report?

a) Gram variable coccobacilli

b) Gram negative coccobacillary organisms

c) Gram negative cocci in chains

d) Wait and see what grows

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