10/11/19

Case Studies

Lars F. Westblade, Ph.D, D(ABMM) (E-mail: [email protected]) Weill Cornell Medicine

SCASM, San Diego

October 26, 2019

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The Wonderful World of Weirdobacter weirdii species!

Lars F. Westblade, Ph.D, D(ABMM) (E-mail: [email protected]) Weill Cornell Medicine

SCASM, San Diego

October 26, 2019

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Disclosures

• Grant/Research support: - Accelerate Diagnostics, Inc. - BioFire Diagnostics, LLC - Hardy Diagnostics

• Advisory Board: - Roche Molecular Systems, Inc.

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Objectives

• List diagnostic features of microorganisms not commonly encountered or recognized in the clinical microbiology laboratory

• Describe their significance and clinical presentation

• Discuss their key antimicrobial susceptibility traits

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What are Weirdobacter weirdii Species?

• Organisms rarely encountered or recognized in the clinical microbiology laboratory à be at the ready!

• Can be difficult to identify using biochemical methods

• Provide opportunity for inane trivia!

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Case 1: Color Me Bad • 11-year-old male from Alabama presents to regional community hospital with gluteal

• Had been standing in stagnant water

• Oral (PO) clindamycin administered as outpatient

2 days

Lesions ~2 cm in diameter

6 Richard et al., 2015 Am J Case Rep 16:740-4 6

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Case 1: Color Me Bad

• Within 24 h developed fever and admitted to local community hospital for surgical drainage of presumed Staphylococcus aureus soft tissue infection

• Intravenous (IV) clindamycin and initiated

• Within 36 h of admission develops dyspnea/hypoxia

• Transferred to PICU at tertiary care medical center

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Case 1: Color Me Bad • Patient intubated for impending respiratory failure and progressive shock

• Physical examination revealed two large, indurated on buttocks

• Multiple pustules on chest, abdomen, and extremities: concern for staphylococcal infection or ecthyma

8 Richard et al., 2015 Am J Case Rep 16:740-4 8

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Case 1: Color Me Bad

• Developed acute respiratory distress syndrome

• Placed on venoarterial extracorporeal membrane oxygenation (ECMO) to maximize cardiac output

• Antimicrobial coverage: vancomycin, clindamycin, nafcillin (for presumed staphylococcal infection)

• Rare Gram-negative rods (GNR) observed in wound Gram stain

• Ceftriaxone added based on wound Gram stain result; later changed to meropenem because of patient’s decompensation

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Case 1: Color Me Bad

• Wound/blood cultures grew a GNR

• Grew on blood agar (BAP) à violet pigmentation

Gram stain: medium sized GNR Violet-colored colonies on BAP

10 Image credit: https://www.cdc.gov/microbenet/ 10

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Case 1: Color Me Bad • Chromobacterium violaceum

• Treated with meropenem, IV ciprofloxacin and IV trimethoprim- sulfamethoxazole (SXT) – all low MIC values (therapy paired to meropenem and ciprofloxacin once antibiotic susceptibility data available)

• Discharged on day 40 on ertapenem (?) and PO SXT

• After ~4 mo of therapy, ESR mildly elevated, continued on PO ciprofloxacin and SXT for a total of 6 mo therapy

• Developed autoamputation of distal end of right thumb and several toes, required skin graft of dorsum of right foot

• Returned to school 4 mo after initial illness, no other permanent sequelae 11 11

Case 1: Color Me Bad

• Diagnosed with chronic granulomatous disease (CGD)

• c.75-76delGT mutation in NCF1 gene (NCF1 encodes a subunit of the phagocyte NADPH oxidase)

• Past medical history of submental abscess at 2 yrs that grew Nocardia spp, supports diagnosis of CGD

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C. violaceum: Clinical Significance

• Inhabits soil/water in tropical/subtropical climates

• Clinical presentation (entry through skin [rarely oral]): - Localized infection (wound infections) - Disseminated infection, sepsis

• Endowed with numerous virulence factors: adhesins, invasins, cytolytic proteins

• Associated with CGD/glucose-6-phosphate dehydrogenase deficiency à high mortality rate

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Chronic Granulomatous Disease

• An inherited immunodeficiency disorder: inability of phagocytes to kill microbes they have ingested (usually discovered early in childhood)

• Recurrent infection (every 3 to 4 yrs) of lung, skin, lymph nodes, and liver (granulomatous inflammation of affected organs)

• CGD infections caused by (catalase POS [environmental] organisms): - Aspergillus spp - Nocardia spp - S. aureus - (Pigmented) Gram-negatives: C. violaceum, Burkholderia cepacia complex, , and

• Defective enzyme in phagocytes - NADPH oxidase complex - results in impaired production of reactive oxygen species (ROS), in phagocytes

• ROS kill microbes à thus lower levels of ROS leads to decreased microbial killing 14

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Relationship between CGD and Catalase- Positive Organisms

Human NADPH oxidase complex - + + NADPH + 2O2 à 2O2 + NADP + H

Human Superoxide dismutase + - 2H + 2O2 à H2O2 + O2

Bacterial Catalase 2H2O2 à 2H2O + O2 15

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Relationship between CGD and C. violaceum in the SE USA

16 Macher et al., 1982 Ann Intern Med 97:51-5 16

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C. violaceum: Microbiology

• Motile GNR, glucose-fermenter (non-lactose-fermenter)

• Facultative anaerobe

• Cultures have almond-like odor (ammonium cyanide)

• Pigmented (~90%) and non-pigmented (~10%) strains

• Oxidase (OX) POS/indole (IND) variable (V) - Non-pigmented strains typically IND POS

• Non-pigmented strains can be confused with Aeromonas spp and Vibrio spp (automated systems can misidentify as Burkholderia pseudomallei)

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C. violaceum: Microbiology

Vibrio spp: Aeromonas spp: - OX, POS - OX, POS - IND, POS - IND, POS - Lysine decarboxylase, POS - Violet pigmentation, NEG (except, A. caviae complex) - TCBS growth, POS - TCBS growth, NEG

C. violaceum: - OX, POS - IND, V - Lysine decarboxylase, NEG - Violet pigmentation, V - Almond-like odor

- TCBS growth, NEG 18 Image credit: Selma Salter, Stamford Health 18

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C. violaceum: Microbiology Aeromonas spp: Vibrio spp: - OX, POS - OX, POS - IND, POS - IND, POS - Lysine decarboxylase, POS - Violet pigmentation, NEG (except, A. caviae complex) - TCBS growth, POS - TCBS growth, NEG

C. violaceum: - OX, POS - IND, V - Lysine decarboxylase, NEG - Violet pigmentation, V - Almond-like odor - TCBS growth, NEG 19 Image credit: Selma Salter, Stamford Health 19

Oxidase Activity of Pigmented Strains: Ascending Paper Chromatography

Inset bottom of filter Filter Paper paper in petri dish

Organism Schmear

Petri dish filled with 2-10 mL oxidase reagent (TMPD)

Dhar and Johnson, 1973 J Clin Pathol 26:304-6 20 Slesak et al., 2009 Ann Clin Microbiol Antimicrob 8:24 20

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Oxidase Activity of Pigmented Strains: Ascending Paper Chromatography - + + Product of reaction (indophenol blue) Filter Paper “wicks” up paper

Petri dish filled with 2-10 mL oxidase reagent (TMPD)

Dhar and Johnson, 1973 J Clin Pathol 26:304-6 21 Slesak et al., 2009 Ann Clin Microbiol Antimicrob 8:24 21

Testing Oxidase Activity of Pigmented C. violaceum Strains

E. coli C. violaceum P. aeruginosa - + + Distilled water (2-10 mL)

Schmear of organism

1% TMPD (oxidase reagent, 2-10 mL)

Schmear of organism

Dhar and Johnson, 1973 J Clin Pathol 26:304-6 22 Slesak et al., 2009 Ann Clin Microbiol Antimicrob 8:24 22

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C. violaceum: Antibiotic Susceptibility

• No established breakpoints for AST results

• High MIC values: polymyxins/some b-lactams

• Low MIC values: fluoroquinolones, tetracyclines, SXT

23 McAuliffe et al., 2015 Am J Trop Med Hyg 92:605-10 23

Color Me Bad: Violacein

• Ethanol-soluble violet pigment

• Two molecules of L-tryptophan à pyrrolidone scaffold

• Role in evading the immune system: - Neutralization of ROS - Induce apoptosis of leukocytes (potential chemotherapy)

• Strong antimicrobial activity against /protozoa 24 Image credit: Lopes et al., 2009 Antimicrob Agents Chemother 53:2149-52 24

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Case 2: Look What the Dog Dragged In • 4-year-old dog (mixed breed)

• Rescue recently imported from Thailand

• Paralyzed hind legs (spinal injury), urinary incontinence

• Urine culture requested because of incontinence

Case Courtesy of K. Deriziotis, J. Moody, D. Peck, R. Franklin-Guild, and A. Thachil College of Veterinary Medicine, Animal Health Diagnostic Center, Cornell University 25 Image credit: http://www.handicappedpets.com/blog/ 25

Case 2: Look What the Dog Dragged In • Urine Culture: - 24 h: >105 pinpoint colonies on BAP and eosin methylene blue agar (EMB) - 48 h: mucoid colonies on BAP and EMB (NLF)

• MALDI-TOF MS (Bruker), Burkholderia thailandensis (≥2.00)

• Culture immediately moved to BSL-3 facility

26 Image credit: Cornell University, AHDC 26

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Case 2: Look What the Dog Dragged In

• “Presumptive B. pseudomallei” NYS DOH PHL Biodefense Laboratory (determined by PCR)

• Confirmed as an atypical strain of B. pseudomallei at CDC

• Atypical characteristics: - Beta-hemolytic on BAP at 48 h (sometimes observed around areas of confluent growth, not around individual colonies) - Catalase NEG – unusual!

• Tier 1 select agent; Federal Select Agent Program

27 https://www.selectagents.gov/ohp-app1.html 27

B. pseudomallei: Clinical Significance

• Agent of human/animal (~165,000 cases yr/worldwide)

• Endemic in SE Asia, N Australia, India, China, Caribbean (Puerto Rico) - tropical areas: case fatality rate estimated as high as 50%!

• Environmental organism, resident in soil/water

• Infection is seasonal, up to 85% of cases during monsoon wet season

• Severe weather events/environmental disturbances have been associated with clusters (e.g., Asian tsunami 2004) 28

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B. pseudomallei: Clinical Significance

29 http://www.promedmail.org/ 29

B. pseudomallei: Clinical Significance

• Acquired through inoculation, inhalation, aspiration and ingestion (contaminated water sources)

• Presentation (incubation period, Av, 9 d [range, 1-21 d] up to many yrs [62 yrs!]): - Acute, localized infection: ulcer, nodule or skin abscesses - Pulmonary infection (common): bronchitis, , cavitary lesions - Acute, bloodstream infection - Chronic suppurative infection: joints, lymph nodes, liver, lung, spleen

• Risk factors: diabetes, renal insufficiency, cystic fibrosis, alcohol excess; zoonotic transmission rare

• Suspect in individuals with travel to endemic area who present with fever of unknown origin or TB-like illness

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B. pseudomallei: Microbiology • Small bipolar staining GNR, glucose non-fermenter

• Culture: - BAP: small, smooth, creamy in first 48 h à further incubation, wrinkled/crinkled colonies - Ashdown agar: pinpoint colonies at 18 h à purple, flat, dry and wrinkled/crinkled colonies after 48 h incubation

Colony Gram stain Growth on BAP Growth on Ashdown agar

31 Image credit: Manual of Clinical Microbiology, 11th Edition 31

Wrinkled/Crinkled Microbes Test B. pseudomallei B. mallei B. thailandensis Pseudomonas (GNR, bipolar (Gram-negative stutzeri staining) coccobacillus) Oxidase + V + +

Motility + 0 + + (# of flagella) (≥2) (≥2) (1) Growth on + V + + MacConkey Growth at 42˚C + 0 + V

Wrinkled/crinkled + 0 + + colonies (>48 h incubation) Arginine + + + 0 dihydrolase Arabinose 0 0 + Unknown assimilation (to the presenter) Polymyxin R R R S susceptibility

Abbreviations: + positive; 0, negative; V, variable; R, resistant; S, susceptible Note, Pseudomonas oryzihabitans is OX NEG, motile, yellow-colored, and polymyxin susceptible 32

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B. pseudomallei: Antibiotic Susceptibility • Intrinsically resistant to: - - 1st and 2nd - - Macrolides - Polymyxins

• Resistant to aminoglycosides (B. mallei susceptible); both B. mallei and B. pseudomallei susceptible to amoxicillin- clavulanate (AMC) - unusual

• Treatment: - Initial intensive therapy: ceftazidime or meropenem - Eradication therapy: SXT or AMC

• Post-exposure treatment: SXT or AMC 33

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B. pseudomallei Antibiotic Susceptibility

Amoxicillin-clavulanate

Colistin

Gentamicin

34 Image credit: Dr. David Dance, Lao-Oxford-Mahosot Hospital-Wellcome Trust Research Unit 34

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Potential for B. pseudomallei in the Southeastern US?

35 Portacci et al., 2017 J Am Vet Med Assoc 250:153-9 35

Potential for B. pseudomallei in the (Southeastern) US?

36 Glass et al., 2006 Int J Syst Evol Microbiol 56:2171-6 36

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Potential for B. pseudomallei in the US?

37 Glass et al., 2006 Int J Syst Evol Microbiol 56:2171-6 37

Case 3: You’re Breaking My Heart!

• 29-year-old male with 18 mo of intermittent fever and night sweats

• Unintentional weight loss, 6 kg

• Tetralogy of Fallot with Blalock shunt at 7 d old

• replacement (PVR), 2006

• Bovine bioprosthesic PVR, 2014 Tetralogy of Fallot (a cause of cyanosis): - Ventricular septal defect • LivaNova 3T Heater-Cooler device - Pulmonary valve stenosis used during surgery in 2014 - Misplaced aorta - Thickened right ventricular wall

38 Image credit: https://www.kidsheartshouston.com/ 38

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Case 3: You’re Breaking My Heart!

• Travel to Pakistan, Thailand, Laos, and Myanmar Transesophageal echocardiography

• Close contact with farm animals in Pakistan

• Consumption of unpasteurized milk in St. Louis, MO

• 3/6 holo-systolic murmur at left upper and lower sternal border Erratically moving echodensity

• Hepatosplenomegaly Also observed severe pulmonary • Blood cultures negative à culture- stenosis (narrowing) and elevated negative (CNE) pulmonary artery pressure

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Case 3: You’re Breaking My Heart!

• Differential diagnosis: - Mycobacterium chimaera - - henselae - Brucella species - HACEK organisms

40 Image credit: https://www.fda.gov/medicaldevices/ 40

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Case 3: You’re Breaking My Heart!

• Patient at risk of sudden cardiac death

• Ideally, treatment should be initiated before surgery to reduce risk of reinfection of prosthetic material

• Serologic testing may be inconclusive and too targeted (slow?)

• Culture and molecular testing of explanted tissue may be required for diagnosis of etiologic agent

• High-throughput sequencing (next-generation sequencing [NGS]) of microbial cell-free DNA in plasma à identify etiologic agent(s) of CNE (The Karius Test)

41 https://www.kariusdx.com/products/karius-test 41

NGS of Microbial cfDNA

Bacteria +750 Fetal DNA

DNA Viruses Transplanted +100 Tissue DNA

Tumor DNA Fungi +300 Pathogen DNA cfDNA in Other plasma Eukaryotes +50

42 Image credit: Karius 42

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Karius Test Result (Within 24-48 h!)

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C. burnetii: Clinical Significance

• Obligate intracellular pathogen, cell wall similar to Gram-negative bacteria

• Etiologic agent of “Q” (Query) fever – first described in Australia, 1935

• Two antigenic phases observed due to phase variation of surface lipopolysaccharide: - Phase I, highly infectious - Phase II, not infectious (spore-like; assists in extracellular survival of the organism)

• Most commonly transmitted through: - Ingestion of infected milk/milk products - Inhalation of birth products or excreta of infected cattle, sheep and goats - Contact with tick feces - Arthropod bites (especially ticks) – exceptional cases

• Clinical presentation: - Asymptomatic infection - Undifferentiated febrile disease (acute or chronic) - Granulomatous hepatitis (acute) - Atypical pneumonia (acute) - Subactute bacterial endocarditis (chronic)

Reimer, 1993 Clin Microbiol Rev 6:193-98 44 Eldin et al., 2017 Clin Microbiol Rev 30:115-90 44

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C. burnetii: Diagnosis • Diagnosis: - Serology: indirect immunofluorescence (reference), complement fixation, ELISA - PCR, clinical specimens - Culture (shell vial, HEL [lung] cell monolayers), clinical specimens - Immunohistochemistry, clinical specimens

• Serology, first-line diagnostic technique: - Elevated phase I IgG titers (≥1:1,800) associated with chronic infection - Elevated phase II IgG titers (≥200) associated with acute infection

• Our patient’s titers:

Antibody Titer IgG Phase I 1:1,048,576 IgG Phase II 1:2,097,152

Reimer, 1993 Clin Microbiol Rev 6:193-98 45 Eldin et al., 2017 Clin Microbiol Rev 30:115-90 45

Case 3: You’re Breaking My Heart!

• Patient was diagnosed with endocarditis

• Started on hydrochloroquine and doxycycline

• Complete resolution of symptoms in 4 wks

• Underwent successful PVR with graft exchange

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Case 3: You’re Breaking My Heart! • Formalin-fixed, paraffin-embedded (FFPE) valve tissue sent to CDC

• IHC negative for C. burnetii

• C. burnetii DNA amplified from FFPE valve tissue

• Genotyping: - Plasma cfDNA (Karius): ST 8 (Group IV) - DNA from FFPE tissue: STs 9, 10, 27, 28, 31 (Group IV)

• STs: - ST 8, goat North America - ST 9, goat Afghanistan (also found in France) 47

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Case 3: You’re Breaking My Heart!

48 Hornstra et al., 2011 PLoS One 6:e26201 48

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Case 3: You’re Breaking My Heart!

United States Pakistan

?

49 Image credit: https://www.wikipedia.org/ 49

Case 3: You’re Breaking My Heart!

Antibody Titer Antibody Titer

IgG Phase I 1:1,048,576 IgG Phase I 1:131,072 8-fold ↓

IgG Phase II 1:2,097,152 IgG Phase II 1:65,536 32-fold ↓

1 month 5 months PVR Surgery

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Case 4: Too Beta to be a “B”!

• A 29-year-old African American woman seen in her 3rd pregnancy: - 1st pregnancy, premature rupture of membranes à spontaneous delivery of a previable infant at 5 months - 2nd pregnancy, spontaneous abortion at 7 weeks

• Had a history of abnormal cervical cytology smears à colposcopy and loop electrosurgical excision procedure

• Vaginorectal culture taken at 39 weeks for Streptococcus agalactiae (GBS)

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Case 4: Too Beta to be a “B”!

Overnight growth on TSAB at 35˚C in 5% CO2

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Case 4: Too Beta to be a “B”!

Colony Gram stain (1,000 × [oil immersion])

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Case 4: Too Beta to be a “B”!

• Catalase: negative

• PYR: negative

• Group B latex typing reagent: weak positive

• What about that zone of b-hemolysis?

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Case 4: Too Beta to be a “B”!

Case 4 GBS

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Case 4: Too Beta to be a “B”!

• Catalase: negative

• PYR: negative

• Group B latex typing reagent: weak positive

• What about that zone of b-hemolysis?

• MALDI-TOF MS (Vitek MS): Streptococcus porcinus

• 16S sequencing: Streptococcus pseudoporcinus

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Case 4: Too Beta to be a “B”!

• Nonhuman isolates à S. porcinus • Human isolates à S. pseudoporcinus • S. pseudoporcinus (Lancefield antigen: E, P, NG1, untypeable) can cross-react with commercial Group B typing reagents • Similar to GBS: - Large colonies, >0.5 mm after 24 h incubation - CAMP factor positive - Hippurate positive - Bacitracin resistant - Isolated from the female genital tract - Susceptible to • May be indistinguishable from GBS on GBS chromogenic agars, however, not detected by GBS molecular assays • Identified by MALDI-TOF MS (Vitek MS and MALDI Biotyper) either as S. porcinus or S. pseudoporcinus (if identified as S. porcinus, human isolates are very likely S. pseudoporcinus)

Suwantarat et al., 2015 J Clin Microbiol 53:3926-30 Gullett et al., 2017 J Clin Microbiol 55:1604-7 Gullett et al., 2017 J Clin Microbiol 55:1973-4 57 Grundy et al., 2019 Am J Obstet Gynecol 220:490.e1-490.e7 57

Case 4: Too Beta to be a “B”! • 1.6% of women colonized in pregnancy (5-6% in all females) • Colonization independently associated with: - Black women - Tobacco use - BMI ≥35 • Compared to women colonized with GBS, women colonized with S. pseudoporcinus more frequently experience preterm premature rupture of membranes (PPRM) or spontaneous preterm birth

• No significant difference in rates of chorioamnionitis, postpartum fever, endomyometritis, or wound infections between GBS and S. pseudoporcinus colonization groups

• Neonates delivered by women colonized with S. pseudoporcinus more frequently admitted to the NICU (but no difference in rates of neonatal sepsis/respiratory distress syndrome between GBS and S. pseudoporcinus carriers)

• Value in reporting S. speudoporcinus colonization in the electronic medical record and indicating its association with PPRM and spontaneous preterm birth Suwantarat et al., 2015 J Clin Microbiol 53:3926-30 Gullett et al., 2017 J Clin Microbiol 55:1604-7 Gullett et al., 2017 J Clin Microbiol 55:1973-4 58 Grundy et al., 2019 Am J Obstet Gynecol 220:490.e1-490.e7 58

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Case 4: Too Beta to be a “B”!

• Patient required Cesarean delivery at gestational age 39.3 wk after failed induction of labor for severe gestational hypertension/preeclampsia

• No premature rupture of membranes à no intrapartum antibiotics administered

• Both patient and infant did well

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GBS vs. S. pseudoporcinus

Characteristic Streptococcus Streptococcus agalactiae pseudoporcinus ATCC 13813T

Hemolysis b b (TSAB) (soft/narrow) (large)

Group B Antigen/Cross- + + Reactivity

LAP + +

V PYR 0 (often NEG)

6.5% NaCl + +

Esculin 0 +

Arginine + +

Hippurate + +

Abbreviations: LAP, leucine aminopeptidase; 0, negative; +, positive; PYR, pyrrolidonyl arylamidase; V, variable 60 Shewmaker et al., 2016 J Clin Microbiol 54:739-44 (modified) 60

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Case 5: It’s Not Easy Being Green

• 30-year-old male sustained traumatic freshwater injury jet-skiing in Missouri (immunocompetent)

• Culture of adipose tissue grew green-colored organism on Sabouraud dextrose agar

Westblade et al., 2015 New Eng J Med 372:982-4 61 Image credit: Brad Ford, University of Iowa 61

Case 5: It’s Not Easy Being Green

10 µm

62 Image credit: Brad Ford, University of Iowa 62

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Meanwhile in Texas…

Colleagues,

We have isolated an organism we are pretty sure is Prototheca but colony morphology doesn't fit. MCM describes colonies as white and yeast-like. This colony is a green as can be on Sab Dex. Microscopically, it appears as an oblong rather than round algae, so I assume it is a P. zopfii. Patient had a water exposure. Does anyone know of a lab that could verify the identity?

Thanks

Bob

Bob Fader, Ph.D. D(ABMM) Section Chief, Microbiology/Virology Scott & White Healthcare Temple, TX 76508 254-724-2714 (office) 254-724-8776 (fax)

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Case 5: It’s Not Easy Being Green

• Both Missouri and Texas isolates identified as Desmodesmus armatus: chlorophyll-containing alga

• Both Missouri and Texas patients required surgical debridement, no antifungals administered

• Both recovered without recurrence of D. armatus infection

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Chlorellosis • Chlorellosis caused by infection with chlorophyllic algae, primarily an infection of animals

• Genus associated with animals: probably Chlorella spp

Clinical material obtained from an ewe Lung tissue obtained from an ewe (ovine chlorellosis) Grocott’s methenamine silver stain

65 Ramírez-Romero et al., 2010 Mycopathologia 169:461-6 65

Human Chlorellosis

• Two organisms described in human infections: - Chlorella spp: endosporulating, round to oval microorganisms, 6 to 9 µm in diameter - D. armatus

• A handful of cases described (4?, including ours)

• Treatment: surgery, antiseptic baths

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Human Chlorellosis Image of lesion PAS-positive cytoplasmic granules in granulomatous lesion (1,500 ×)

TEM of degenerated, collapsed algal cell containing membranous profiles remnant of a chloroplast, CP (28,440 ×)

67 Jones et al., 1983 Am J Clin Pathol 80:102-7 67

Case 2: LUNGKNOWN

• A 59-year-old Moroccan man with history of metastatic urothelial cell carcinoma à presents with fever, shortness of breath and chest pain

• Two years prior he had received intravesicular Mycobacterium bovis BCG therapy for urothelial carcinoma

• Negative tests: - Blood cultures - serogroup 1, Streptococcus pneumoniae, Histoplasma capsulatum antigens - Serum galactomannan and cryptococcal antigen

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Case 2: LUNGKNOWN

Computed Tomographic Scan of Chest Demonstrating Cavitary Lung Nodules

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Case 2: LUNGKNOWN

Acid-Fast Stain of Lung Core Biopsy (Kinyoun; 1,000 ×)

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Case 2: LUNGKNOWN

• Differential?

• Full acid-fast (Kinyoun) positive organisms: - Mycobacterium species - M. bovis BCG

• Modified acid-fast positive organisms: - Gordonia species - Nocardia species - Tsukamurella - Rhodococcus equi

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Case 2: LUNGKNOWN

• QuantiFERON TB Gold test, negative

• PCR for M. tuberculosis complex on expectorated sputum, negative

• Lung biopsy: growth observed on BCYE on day 6

Image: Anne Norris, NYPH-WCMC 72

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Case 2: LUNGKNOWN

Colony Gram Stain, Carbol Fuchsin Counterstain (1,000 ×)

Image: Amy Roberston/Rebecca Marrero, NYPH-WCMC 73

Case 2: LUNGKNOWN

• Colony Gram stain, Gram-negative rods (stain better with carbol fuchsin than safranin)

• Colony modified and full acid-fast stains, negative

• MALDI-TOF MS à Legionella micdadei

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Case 2: Legionella micdadei

• Acid-fast (modified [Fite] and full [Kinyoun]) positive in fresh and formalin-fixed tissue

• Modified acid-fast staining protocols optimal

• Acid-fast staining property lost when cultivated on agar

• Characteristics: - OX: POS - Motility: POS - UV autofluorescence (366 nm): negative

Koneman’s Color Atlas and textbook of Diagnostic Microbiology, 7th Ed Wolters Kluwer Manual of Clinical Microbiology, 11th Ed ASM Press 75

Case 2: Legionella micdadei

• Mycobacteria à beaded acid-fast bacilli

• L. micdadei à small coccobacillus

M. tuberculosis: full acid-fast stain L. micdadei: full acid-fast stain 1,000 ×: beaded rods 1,000 ×: small coccobacilli

Image: https://en.wikipedia.org/wiki/Ziehl%E2%80%93Neelsen_stain 76

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Case 2: Legionella micdadei

• After identification of L. micdadei patient treated with 3-week course of levofloxacin

• Patient’s condition improved clinically

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