OSCE NL3: Staining

Narachai Julanon, MD
Department of internal medicine

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 Outline
• Gram stain
• Acid fast, modified acid fast stain
• Wright stain
• Fresh smear
• Stool exam

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Proper field in Gram stain

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 Description
• Specimen
• Staining method
• Cell: Wbc, epithelium ***
• Organism

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 Question
• Description
• Causative organism
• Diagnosis/disease
• Treatment

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1. A 50-year-old Man presented with right ankle arthritis 1 wk

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 Answer
• Synovial fluid Gram stain
– Numerous PMN
– Gram positive cocci in cluster (grape-like)
• Diagnosis: Staphylococcal septic arthritis at
right ankle (Most likely Staphylococcus aureus)
• Treatment:
– Cloxacillin 1 g IV q 6 hr 4 – 6 wk
– Drainage

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2. A 70-year-old woman presented fever + CVA tenderness 2 d

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 Answer
• Urine Gram stain
– Numerous PMN
– Gram positive cocci in chain
• Diagnosis: acute pyelonephritis from
Enterococcus spp.
• Treatment:
– Ampicillin 1 g IV q 6hr duration 1-2 wk

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 Gram positive cocci in chain
• Streptococcus pyogenes (GAS): pharyngitis,
skin infection
• Streptococcus agalactiae (GBS):
chorioamnionitis, neonatal sepsis, meningitis
• Enterococcus spp.: bacteremia, IE, UTI,
peritonitis
• Streptococcus suis: meningitis

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3. A 30-year-old man presented fever + headache 2 d,
PE: neck stiffness

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 Answer
• CSF Gram stain
– Few PMN
– Gram positive lancet-shaped diplococci with
capsule
• Diagnosis: Streptococcus pneumoniae
meningitis
• Treatment:
– Ceftriaxone 2 gm IV q 12 hr x10 – 14 d

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 Streptococcus pneumoniae
• Otitis media
• Sinusitis
• Meningitis
• Pneumonia

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4. A 30-year-old man presented fever + right leg swelling 2 d,
PE: crepitus at right leg

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 Answer
• Pus from right leg Gram stain
– Numerous PMN
– Gram positive bacilli, boxcar-shaped
• Diagnosis: gas gangrene (myonecrosis) right
leg from Clostridium perfringens
• Treatment:
– Surgical debridement
– PGS 4 MU IV q 4hr + clindamycin 900 mg IV q 8hr
7 – 14 d

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5. A 30-year-old man presented with COPD exacerbation,
PE: fever, crepitation RLL

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 Answer
• Sputum Gram stain
– Adequate sputum
– Gram negative diplococci
• Diagnosis: pneumonia RLL from Moraxella
catarrhalis
• Treatment:
– Ceftriaxone 2 gm IV OD x7 d
– Amoxicillin-clavulanate 1.2 gm IV q 8 hr x7 d

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6. A man, 30 year presented with dysuria 5 d

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 Answer
• Pus from urethra Gram stain
– Numerous PMN
– Intracellular Gram negative diplococci, kidney-
shaped
• Diagnosis: Gonorrhea
• Treatment:
– Ceftriaxone 250 mg IM single dose
– Doxycycline (100) 1x2 PO pc 7d

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 Gram negative diplococci
• Sputum (community): Moraxella catarrhalis
• Sputum (nosocomial): Acinetobacter
baumannii
• Urethral discharge: Neisseria gonorrhoeae
• CSF: Neisseria meningitidis

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A 30-year-old man, during hospital admission, presented with
fever + cough, PE: crepitation RLL

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Acinetobacter baumannii pneumonia

• Imipenem, meropenem
• Ampicillin/sulbactam
• Cefoperazone/sulbactam
• Colistin

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7. A 30-year-old man presented with fever + cough 5 d
PE: crepitation RLL

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 Answer
• Sputum Gram stain
– Adequate sputum
– Gram negative coccobacilli
• Diagnosis: pneumonia RLL from Haemophilus
influenzae
• Treatment:
– Ceftriaxone 2 gm IV OD x7 d

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8. A 30-year-old man presented with fever + right wrist
arthritis 10 d

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 Burkholderia pseudomallei
• Synovial fluid Gram stain
– Moderate PMN
– Gram negative bacilli, bipolar staining (safety pin)
• Diagnosis: right wrist septic arthritis from
Burkholderia pseudomallei
• Treatment:
– Ceftazidime 2 g IV q 8 hr x2 wk then
– Cotrimoxazole SS (TMP/SMZ 80/400) 3x2 PO pc
after D14 until 20wk
BW <40 40 – 60 >60
Bactrim SS 2x2 3x2 4x2 30
 Melioidosis
• Pneumonia
• Hepatosplenic
• Bacteremia
• Septic arthritis

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9. A 50-year-old man, U/D alcoholism presented with fever +
cough 5 d, PE: crepitation right lung

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 Answer
• Sputum Gram stain:
– Numerous PMN
– Large Gram negative bacilli with thick capsule
• Diagnosis: right lung pneumonia due to
Klebsiella pneumoniae
• Treatment:
– Ceftriaxone 2 gm IV OD x7 d

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A 50-year-old man with UTI

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 Enterobacteriaciae
• Escherichia
• Klebsiella
• Proteus
• Serratia
• Shigella
• Salmonella
• Enterobacter

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10. A 50-year-man presented with cavitary lung lesion 10 d

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 Answer
• Sputum Gram stain:
– Numerous PMN
– Mixed pleomorphic gram positive and gram
negative organisms
• Diagnosis: anaerobic lung abscess
• Treatment
– Clindamycin 150 – 300 mg IV q 6 hr x4 – 6 wk
– Amoxicillin/clavulanate 1.2 gm IV q 8 hr x4 – 6 wk

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11. A 50-year-old man presented with chronic cough

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 Answer
• Sputum acid fast staining
– Short rod, bead-liked acid fast bacilli
• Diagnosis: pulmonary tuberculosis, smear positive
• Treatment: 2IRZE/4IR
– Isoniazid (100) 3 tab PO hs
– Rifampicin (450) 1 tab PO hs (BW <45) or
Rifampicin (300) 2 tab PO hs (BW >45)
– Ethambutol (400) 2 tab PO hs
– Pyraziamide (500) 3 tab PO hs
– B6 (50) 1 tab PO OD

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12. A 30-year-old SLE woman presented with chronic cough

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 Answer
• Sputum modified acid fast staining
– Moderate PMN
– Modified acid fast beaded right-angled branching
filamentous organism
• Diagnosis: pulmonary nocardiosis
• Treatment
– Trimethoprim/Sulfamethoxazole 15 MKD TMP
IV/PO x 4 wk then 10 MKD x 3 – 6 mo*

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Gram positive branching filamentous organism?

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 Gram positive branching filamentous
organisms
• Nocardia: mAFB +ve
• Actinomyces: mAFB –ve

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13. A 30-year-old SLE woman presented with cough

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 Answer
• Sputum (any staining)
– Inadequate
– Seen Strongyloides stercoralis larva
• Diagnosis: disseminated strongyloidisis
• Treatment
• Oral Ivermectin 200 µg/kg OD until symptom resolve or
larvae undetectable for at least 2 weeks
• Ceftriaxone 2 g IV OD

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14. Pus from central line

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 Answer
• Pus Gram stain
– Numerous PMN
– Budding yeast with pseudohyphae
• Diagnosis: suppurative candidal
thrombophlebitis
• Treatment
– Off central line
– Fluconazole 400 mg IV OD 14 d

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15. HIV with lymphadenopathy, pancytopenia

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 Answer
• Bone marrow aspiration Wright stain
– Seen encapsulated budding yeast
• Diagnosis: disseminated cryptococcosis
• Treatment
– Induction: Amphotericin B 0.7 mg/kg IV OD +
fluconazole (200) 2x2 PO x2 wk then
– Consolidation: fluconazole (200) 2x1 PO x8 wk then
– Maintenance: fluconazole (200) 1x1 PO until CD4 >100
x6 mo

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 Cryptococcal meningitis in HIV,
Thailand
• Induction: Amphotericin B 0.7 mg/kg IV OD +
fluconazole (200) 2x2 PO x2 wk then
• Consolidation: fluconazole (200) 2x1 PO x8 wk
then
• Maintenance: fluconazole (200) 1x1 PO until
CD4 >100 x6 mo

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 Histoplasma capsulatum
• Small budding yeast, intracellular and
extracellular, grape-like

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 Penicillium marneffei
• Small oval, round, sausage-shaped yeast with
central clear septum (binary fission)

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 Treatment of histoplamosis and
penicillosis in HIV
• Induction: amphotericin B 0.7 MKD IV x 2wk
• Consolidation: itraconazole (100) 2x2 PO x 10
– 12 wk
• Secondary prophylaxis: itraconazole (100) 2x1
PO

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 These 3 organisms in HIV
• BM involvement
• Lymphadenopathy
• Hepatosplenomegaly
• Umbilicated papules

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A 50-year-old-man with lung mass

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Pulmonary cryptococcosis in non-HIV

• Mild-to-moderate severity: fluconazole 400

mg/d 6 – 12 mo
• Severe: as CNS disease

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16. HIV with diarrhea, stool mAFB

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 Answer
• Stool modified acid fast stain
– Seen modified acid fast oval shaped oocyst
• Diagnosis: Isosporiasis
• Treatment
– Trimethoprim/Sulfamethoxazole SS 2 x 4 PO 4 wk
(HIV)

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 HIV with diarrhea
• Infection
– Parasite: strongyloidiasis
– Protozoa: Isospora, cryptosporidium, cyclospora
– Bacteria: TB, MAC, salmonellosis
• ARV side effect

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 Differentiate cryptosporidium
from cyclospora
Topic Cryptosporidium Cyclospora
parvum cayetanenesis
Size 5 micron 10 micron
Sporulated Yes No
Border Smooth Rough
On Bactrim Likely Less likely
Hepatobiliary Yes No
involvement

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17. A 30-year-old man with fever 7 d after returning from
Kanchanaburi

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 Answer
• Peripheral blood smear
– Rbc
• Infected Rbc are same in size with normal Rbc
• Seen trophozoite (signet-ring form), double infection,
double chromatin dot, accole form
• Infected rate (>5% = hyperparasitemia)
• DIC?
– Wbc
– Plt
• Plasmodium falciparum infection
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 Plasmodium falciparum

Ring trophozoite Schizont Gamete

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Treatment of P. falciparum

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Artesunate 50 mg
Mefloquine 250 mg
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Treatment of P. falciparum

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 Plasmodium vivax
Amoeboid Schizont Gamete
trophozoite

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Treatment of Plasmodium vivax

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18. A 30-year-old man with Gut obstruction

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 Ascaris lumbricoides eggs
• Describe
– Unfertile egg: Elongated Egg with external
mammillated layer
– Fertile egg: Rounded Egg with external
mammillated layer
– Decorticated egg: Outer layer is absent
• Diagnosis: Ascariasis
• Treatment: Albendazole (200) 2x1 PO PC x 3d

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19. A 30-year-old man with rectal prolapse
Rectal prolapse
Stool wet smear & iodine

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 Trichuris trichiura egg
• Describe
- Barrel-shaped, thick-shelled and a pair of polar
plugs at each end
• Diagnosis: Trichuriasis
• Treatment: Albendazole (200) 2x1 PO PC x 3d

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20. A 30-year-old man with iron deficiency anemia
Iron deficiency anemia
Stool wet smear

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 Hookworm egg
(Ancylostoma and Necator)
• Describe
– Thin-shelled, colorless egg
– Rhabditiform (L1) larvae
• Diagnosis: hookworm infestation
• Treatment: Albendazole (200) 2x1 PO PC x 3d

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21. A 30-year-old man with malabsorptive diarrhea
Malabsorption syndrome
Stool wet smear

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 Capillaria philippinensis egg
• Describe
– Barrel-shaped egg with striated shell and 2
inconspicuous polar prominences
• Diagnosis: Capillariasis
• Treatment: Albendazole (200) 2x2 PO PC x 10d

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22. A 30-year-old man with health check up

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 Opisthorchis viverrini egg
• Describe
– Oval-shaped egg with operculum
• Diagnosis: opisthorchiasis
• Treatment: praziquantel (600) 2x3 PO x2 d

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23. A 30-year-old man with diarrhea

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 Taenia egg
• Describe
– Spherical-shaped egg with thick striate wall
• Diagnosis: Taeniasis
• Treatment: praziquantel (600) 1 tab PO single
dose

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Beware!!! Combined

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24. A 30-year-old man with genital ulcer

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 Answer
• Tzanck smear from base of lesion
– Moderate PMN, Rbc
– Multinucleated giant cell
• Diagnosis: genital herpes simplex type 2
infection
• Treatment: acyclovir (200) 1x5 PO PC x 7d

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24. A 30-year-old man with itchy rash

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 Answer
• KOH preparation from skin lesion
– Septate hyphae with arthrospore
• Diagnosis: dermatophytosis
• Treatment: topical azole

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A 30-year-old man with oral lesion

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 Answer
• KOH preparation from oral lesion
– Budding yeast with pseudohyphae
• Diagnosis: oral candidiasis
• Treatment: topical azole

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24. A 30-year-old man with rash

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24. A 30-year-old man with rash

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 Answer
• Methylene blue scotch tape technique from
skin lesion
– Short hyphae with blastospore (spaghetti and
meatball)
• Diagnosis: pityriasis versicolor
• Treatment: topical azole

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25. A 45-year-old cirrhotic man presented fever +
hemorrhagic bleb left forearm 2 d

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 Answer
• Describe
- Few PMN
- Curved, gram negative bacilli
• Diagnosis: NF due to Vibrio vulnificus.
• Treatment:
– Ceftazidime 2 gm v q 8 hr or
– Ciprofloxacin 400 mg v q 12 hr x 10-14 day

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26. A 30-year-old SLE woman presented with cough

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 Answer
• Sputum Wright stain:
– PMN
– Dichotomous branching septate hyphae
• Diagnosis: invasive pulmonary aspergillosis
• Treatment: voriconazole

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27. A 30-year-old diabetic woman presented with sinusitis
and progressive drowsiness

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 Answer
• Sputum Wright stain:
– PMN
– Right angle board non-septate hyphae
• Diagnosis: rhinocerebral mucormycosis
• Treatment:
– Debridement
– Amphotericin B

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28. A 30-year-old woman with vesiculobullous lesions

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 Answer
• Lesion Wright stain:
– Round cell with perinuclear halo (acantholytic cell)
• Diagnosis: suspected pemphigus vulgaris

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