Supplementary Appendix

This appendix has been provided by the authors to give readers additional information about their work.

Supplement to: Kenyon C, Bonorchis K, Corcoran C, et al. A dimorphic causing disseminated in South Africa. N Engl J Med 2013;369:1416-24. DOI: 10.1056/NEJMoa1215460 Supplementary data: Emmonsia sp, a causing disseminated infection in

South Africa

Table of Contents List of investigators ...... 2 S1: Detailed description of laboratory methodology ...... 2 S2: Description for Emmonsia sp...... 7 Figure S1: Combined deep fungal diagnosed on skin biopsy and dimorphic fungi cultured in the microbiology laboratory at Groote Schuur Hospital 2003 to 2011 ...... 8 Figure S2: Peripheral blood smear...... 9 Figure S3: Histology of a skin punch biopsies from patients with emmonsiosis...... 10 Figure S4: Phylogenetic analyses of Emmonsia and related genera ...... 12 Table S1: Clinical, radiological, histological and microbiological features of 13 HIV-infected patients with Emmonsia sp. infection ...... 15 Table S2: Minimum inhibitory concentrations of nine drugs for Emmonsia sp...... 26 References ...... 27

!

! 1! List of investigators !

Chris Kenyon, Kim Bonorchis, Craig Corcoran, Graeme Meintjes, Michael Locketz, Rannakoe

Lehloenya, Hester F. Vismer, Preneshni Naicker, Hans Prozesky, Marelize van Wyk, Colleen

Bamford, Moira du Plooy, Gail Imrie, Sipho Dlamini, Andrew M. Borman, Robert Colebunders,

Cedric Yansouni, Marc Mendelson and Nelesh P. Govender

S1: Detailed description of laboratory methodology

Specimen processing

A small portion of the skin biopsy specimen (n=9) was crushed with a sterile tissue crusher and inoculated onto brain heart infusion agar (BHIA) (Diagnostic Media Products (DMP), National

Health Laboratory Service, Sandringham, South Africa) incubated at 35ºC and Sabouraud dextrose 4% agar (SDA) with and without amikacin (DMP) incubated at 25ºC and 35ºC for 42 days. Bone marrow (n=2) and blood samples (n=5) were inoculated into standard aerobic and

Myco/F-Lytic BACTEC 9240 bottles (Becton Dickinson Diagnostics, Franklin Lakes, NJ) and incubated aerobically at 35°C for up to six weeks. Positive BACTEC cultures were processed by aspirating an aliquot of sample for Gram stain and inoculating the sample onto SDA. Cultures were examined microscopically using lactophenol cotton blue and Gram stains. Four slide cultures per strain were prepared with SDA and incubated at 25ºC. Unstained preparations were examined under light microscopy after 7, 14, 21 and 30 days. Isolates were inoculated onto SDA at 25ºC, 30ºC and 37ºC and BHIA at 37ºC and 40ºC to determine growth rates, conversion to the

! 2! phase and presence or formation of adiaspores. Plates were examined daily for 3 weeks and then weekly.

Broth microdilution determination of minimum inhibitory concentrations (MICs) of the yeast phase of Emmonsia sp

MICs were determined according to CLSI methodologies (CLSI M27-A3) (6) in round-bottomed

96-well plates (TREK Diagnostic Systems, Inc., Cleveland, Ohio, USA). Yeast suspensions were adjusted to final concentrations of 2.5 x 103 CFU/ml. Inoculated plates were incubated for up to

7 days at 35 oC. MICs were read as the concentration of drug that elicited 100% inhibition of growth () or significant (approx. 50 %) inhibition of growth compared with a drug-free control (itraconazole, fluconazole, voriconazole, posaconazole, flucytosine, micafungin, anidulafungin and caspofungin). The quality control isolates Candida parapsilosis

ATCC 22019 and Candida krusei ATCC 6258 were run in parallel on all days of testing.

DNA sequence-based methods

DNA sequence-based procedures were conducted by four investigators (K.B, C.C., M.V.W. and

A.M.B.). The internal transcribed spacer region (ITS) including the 5.8S rRNA operon was amplified with the ITS 1 and ITS 4 primer pair. A part of the 28S rDNA region was amplified with the LR5 and

LROR primer pair. The following primer pairs were employed for the amplification of portions of the actin

(ActinF-GACAATGGATCCGGTATGTG and ActinR-ACATAGCAGAGCTTCTCCTTG), b-tubulin

(BTUBF-CTCCAAACCGGCCAATGCG and BTUBR-GAGAGTCCGCATGCAGATATC) and PRP8

(PRP8F-GCCAAAGGAACACAGCTGCTTCG and PRP8R-GCTGAGGATTCAGAAAGAGG) genes.

Purified amplicons were subjected to a sequencing PCR. The samples were sequenced in a 3130

! 3! Sequencer (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA). Sequences were subjected to Blast analyses in GenBank (www.ncbi.nlm.nih.gov) for identification. Aligned sequences were analyzed in PAUP 4.0b (Swofford DL (2002) PAUP*. Phylogenetic Analysis Using

Parsimony (*and other methods). Version 4. Sunderland, Massachusetts: Sinauer Associates).

Procedures were conducted by investigators (K.B., C.C., M.V.W. and A.M.B.) at four laboratories.

Laboratories 1,2 and 3: (K.B., C.C. and A.M.B): Fungal DNA was extracted from fungal cultures by the Qiagen QIAmp DNA mini kit (Qiagen, Hilden, Germany) using the tissue extraction protocol. An in-house broad-range PCR was performed using pre-published primers flanking the internal transcribed spacer region 2 (ITS-2) found within the fungal ribosomal RNA gene complex (1). Amplified products were sequenced and DNA sequences were compared with those available in the GenBank, EMBL, and DJB database using the gapped BLASTN 2.0.5 program.

Multiple-sequence alignment was performed with CLUSTAL W, and percent similarity was calculated. This PCR assay was validated by testing 28 different fungal species, which had been fully characterized by a reference laboratory, and sent to our laboratory as part of a proficiency testing scheme. The identity of the organism by the described molecular method was the same as the identification provided by the reference laboratory for all isolates; phenotypic methods by laboratory 1 yielded the incorrect identification for two organisms (personal communication,

K.B.)

! 4! Laboratory 2 (M.V.W): The ZR Fungal/Bacterial DNA MiniPrep™ extraction kit (Zymo, Irvine,

CA, USA) was used to obtain high quality DNA. A positive control of either (ATCC 34875, ATCC 32608) or (ATCC 90028) was included in all procedures. The internal transcribed spacer region (ITS) including the 5.8S rRNA operon was amplified with the ITS1 and ITS 4 primer pair (1). The 28S rDNA region (large sub-unit) was amplified with the LR5 and LROR primer pair (1). PCRs for both gene regions were made in 25

µL reactions consisting of 2-9 ηg DNA, 1U GoTaq® Hot start Polymerase (Promega, Madison,

USA), 1x GoTaq® Hot start buffer, 0.1mM dNTPs, 1.6mM MgCl2, 0.5 µM forward and 0.5 µM reverse primer. The PCR conditions were as follows; 96 ºC for 5 min, followed by 30 cycles of

94 ºC for 30 s, 55 ºC for 30s and 72 ºC for 45 s, followed by an extension step of 72 ºC for 7 min and placed on hold at 4 ºC. These amplicons were run on a 1 % Agarose (SeaKem®, Lonza,

USA) gel and visualised with UV fluorescence on a gel doc system (Vacutec, USA) together with a 100 bp ladder (Fermentas, USA). Positive amplicons were purified using 6 % Sephadex

G-50 with columns (Sigma Aldrich, USA). Purified amplicons were subjected to a sequencing

PCR; reactions consisted of the following; 2 µl 3.1 Big Dye, 1 µl 5x Buffer, 8 µl template, 1 µl

0.5 µM either forward or reverse primer, 3 µl sterile water. Sequencing PCR conditions consisted of 25 cycles of 95 ºC for 10s, 55 ºC for 15s and 60 ºC for 4 min. Sequencing PCR products were cleaned with 6 % Sephadex in columns. The samples were sequenced in a 3130 Sequencer

(Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA). Electrophenograms were inspected in Chromas Lite 2.01 (www.technelysium.co.au). Sequences were subjected to

Blast analyses in GenBank (www.ncbi.nlm.nih.gov) for identification. Similar sequences were downloaded and aligned with MAFFT (www.genome.jp/tools/mafft) (2). Aligned sequences were manually inspected in MEGA 4 (www.megasoftware.net) (3). Aligned sequences were

! 5! analysed in PAUP 4.0b (4). Settings in PAUP were as follows; Missing characters were treated as a fifth characters, stepwise addition, simple sequence addition, one trees was held at each step, the branch-swapping algorithm was set to tree-bisection-reconnection, with steepest descent option not in effect, branches were collapsed if the maximum branch length was zero, Multrees was in effect, topological constraints were enforced and Arthroderma benhamiae was treated as the out-group taxon.

Scanning electron microscopy

A cultured isolate (from case 4) was fixed by flooding with 2.5% glutaraldehyde in 0.2M phosphate buffer. Pieces of agar on which the fungus was growing, were cut out and routinely processed for scanning electron microscopy (rinsed in buffer, post-fixed for 2hrs in 1% Osmium tetroxide, dehydrated in an ethanol series, critical point dried, mounted on stubs, coated with gold-palladium), and viewed on an FEI QuantaTM scanning electron microscope.

! 6! S2: Description for Emmonsia sp.

On Sabouraud agar at 25ºC, colonies were white to pale brown, glabrous at first, becoming powdery and wrinkled or cerebriform with age. Reverse pigment was non-diffusible, tan at first, becoming darker brown with age. Microscopically, hyaline subglobose conidia (1-2 x 1.5-2.5

µm) were borne singularly at the ends of narrow pedicles (<1 µm) which were arranged either singularly or in groups around swollen vesicles at right angles along the hyphae or terminally.

Conidia were slightly flattened along their longitudinal axis, smooth under the light microscope but conspicuously tuberculated under SEM. Cultured at 37oC on brain heart infusion agar, small, smooth grey-brown heaped shiny colonies were formed, with globose to oval, thin-walled, hyaline yeast cells (2 to 4 µm), budding on multipolar narrow bases. In , similar yeast cells were observed.

Type material living isolates of Emmonsia sp. NCPF 4164 are deposited in the National

Collection of Pathogenic Fungi (NCPF) housed at the HPA Mycology Reference Laboratory,

Myrtle Rd. Bristol UK.

! 7! Figure S1: Combined deep fungal infections diagnosed on skin biopsy and dimorphic fungi cultured in the microbiology laboratory at Groote Schuur Hospital 2003 to 2011 (n=76). The change in profile of diagnoses after the introduction of the broad-range fungal PCR assay at the end of 2008 is apparent. (There were 23 cases in the “Other fungal disease” category. A firm diagnosis was made in 18 of these: (7), Madura Foot (5), (2), (2), (1), dermal infection (1)).

Other fungal disease

16 Culture-confirmed emmonsiosis

14 Culture-confirmed

Histopathological diagnosis of histoplasmosis 12

10

8 Number of cases Number

6

4

2

0 2003 2004 2005 2006 2007 2008 2009 2010 2011 Year

! 8! Figure S2: Peripheral blood smear showing neutrophils with multiple phagocytosed yeast- like structures (arrow) from case 13 (Wright-Giemsa staining x 1000).

Photo courtesy of Dr William Van Schalkwyk (NHLS, Cape Town)

! 9! Figure S3: a) Histology of a skin punch biopsy from a patient who developed emmonsiosis whilst on antiretroviral therapy, showing a suppurative granulomatous infiltrate in the superficial dermis, comprising sheets of vacuolated macrophages and small collections of neutrophils (haematoxylin and eosin stain, bar = 0.1 mm). b) Histology of the same skin biopsy showing scattered aggregates of small round . Some are within cytoplasmic vacuoles of macrophages and some show narrow-budding (Grocott silver stain, bar = 0.02 mm). c) Typical histology of a skin punch biopsy from a patient not on antiretroviral therapy, showing focal karyorrhectic debris in the superficial dermis, with minimal associated inflammation (haematoxylin and eosin stain, bar = 0.05 mm). d) Histology of the same skin biopsy showing scattered small round yeasts with occasional narrow-based budding present in the dermis (Grocott silver stain, bar = 0.05 mm).

! 10! a! b!

c! d!

! 11! Figure S4: Phylogenetic analyses of Emmonsia and related genera. Panels A, B, C, D and E show the phylogeny based on the Internal Transcribed Section (ITS), Large Subunit (LSU), Beta tubulin (BT), Intein PRP8 (PRP8) and Actin genes respectively. A total of 586, 358, 776, 654 and 524 positions were analyzed for ITS, LSU, PRP8, BT and Actin respectively. The datasets were aligned with MAFFT v. 6 (settings were as follows: Q-INS-i, Scoring matrix = 1PAM/K=2, Gap opening penalty = 1.53, offset value = 0, Threshold score = 39 {E=8.4e-11). Alignments were also checked manually after MAFFT alignments were made. The structure of the five phylogenetic trees presented here were inferred with the use of the maximum parsimony method. PAUP4.0b10 was utilized to obtain the bootstrap results (indicated on the branches of the trees - not in brackets). Gaps were treated as a fifth character while trees were obtained via the heuristic search option with a stepwise addition of 1000 replicates. MrModeltest2 was utilized to obtain the models for each gene region (ITS = GTR+G, LSU = GTR+I, BT = HKY+I, PRP8 = HKY+G and Actin = GTR+I) that was included in the Bayesian analyses (MrBayes). One thousand trees were discarded as the burin-in for the PRP8 and actin trees and two thousand for the other three datasets. The Bayesian results are included in brackets behind the bootstrap values. (A more detailed description of the methodology is provided in supplementary box S1). In all five trees, the clinical cases described in this report from South Africa grouped together in a well-supported clade (JX398288-298). Sequences are identified by their GenBank accession numbers. Scale bars correspond to the number of substitutions per site. Sequences obtained from GenBank are known by their accession numbers while those with a NCPF number were obtained from the National Collection of Pathogenic Fungi, UK. Sequences for Paracoccidioides, Blastomyces and were obtained from publically-available whole genome sequences for these organisms. The EMBL accession numbers for the 5 loci for NCPF strains 4164, 4236, 4289 and 4091 are HF 563662 through HF 563681.

! 12!

a! JX398292 Emmonsia sp. TYPE NCPF4164 b! JX398294 JX398299 JX398289 JX398298 JX398299 JX398297 JX398297 JX398296 JX398296 JX398295 JX398295 JX398294

73 JX398290 JX398293

JX398293 JX398292

JX398298 100(100) JX398291

Emmonsia sp. TYPE NCPF4164 75 JX398290

(93) JX398291 JX398289

JX398288 JX398288

Emmonsia pasteuriana TYPE NCPF4236 Emmonsia pasteuriana TYPE NCPF4236

Blastomyces dermatitidis ACII01000000 100(100) Emmonsia parva NCPF4289

81(93) Blastomyces dermatitidis ACBU01000000 Histoplasm capsulatum AAJI01000000

Emmonsia sp NCPF4091 89(95) Blastomyces dermatitidis ACBU01000000

100(98) AAJI01000000 100(98) Blastomyces dermatitidis ACII01000000

Histoplasma capsulatum ABRJ01000000 Emmonsia sp NCPF4091

Emmonsia parva NCPF4289 Histoplasm capsulatum ABRJ01000000

Paracoccidioides lutzii ABKH01000000 Paracoccidioides lutzii ABKH01000000

Paracoccidioides brasiliensis ABKI01000000 10 Paracoccidioides brasiliensis ABKI01000000 1

! !

Emmonsia sp. TYPE NCPF4164 JX398290 (96) JX398299 d! c! JX398299 JX398293 JX398296

JX398288 JX398295

JX398298 JX398291

JX398296 JX398297 100(94) JX398295 JX398298 99(99) JX398294 JX398288

JX398292 JX398294 100(100) JX398291 100(100) JX398293

JX398290 JX398292 73(89) (78) JX398289 JX398289

JX398297 Emmonsia sp. TYPE NCPF4164

Emmonsia pasteuriana TYPE NCPF4236 Emmonsia pasteuriana TYPE NCPF4236 100(89) Blastomyces dermatitidis ACII01000000 100(100) Histoplasma capsulatum ABRJ01000000 100(100) 100(100) 100(100) Blastomyces dermatitidis ACBU01000000 Histoplasma capsulatum AAJI01000000

(100) Emmonsia sp NCPF4091 76(100) Emmonsia sp NCPF4091

Histoplasma capsulatum AAJI01000000 Emmonsia parva NCPF4289 100(100)

Blastomyces dermatitidis ACII01000000 Histoplasma capsulatum ABRJ01000000 100(100) 73 Blastomyces dermatitidis ACBU01000000 Emmonsia parva NCPF4289

Paracoccidioides lutzii ABKH01000000 Paracoccidioides lutzii ABKH01000000

Paracoccidioides brasiliensis ABKI01000000 Paracoccidioides brasiliensis ABKI01000000 10 10

!

! 13! Emmonsia sp. TYPE NCPF4164 e! (97) JX398292

JX398291

JX398288

JX398299

JX398298

100 (70) JX398296

JX398295

JX398294

82 (100) JX398293

JX398290

74 JX398289

93 (97) JX398297

Emmonsia pasteuriana TYPE NCPF4236

Emmonsia parva NCPF4289 100 (75)

Histoplasma capsulatum ABRJ01000000 100 (99)

Histoplasma capsulatum AAJI01000000 84 Blastomyces dermatitidis ACII01000000 100 (100)

Blastomyces dermatitidis ACBU01000000

Emmonsia sp NCPF4091

Paracoccidioides lutzii ABKH01000000

Paracoccidioides brasiliensis ABKI01000000 1

! 14! Table S1: Clinical, radiological, histological and microbiological features of 13 HIV-infected patients with Emmonsia sp. infection Case Demographic Background, clinical presentation, laboratory and radiology findings Culture of Histology of skin biopsy Treatment and outcome number information Emmonsia sp.

1. 28-year-old • Background: HIV-infected; CD4+ T-lymphocyte count = 40 cells/µl; Positive (skin Groups of PAS stain- • Amphotericin B for 14 days followed

Black male culture-confirmed pulmonary with good clinical and biopsy) positive narrow-based by itraconazole

microbiological response to 3 months of therapy budding yeasts (3 to 7µm in • 3TC/D4T/EFZ

• History: Two-week history of lesions on face (lesions later spread to the diameter) in dermis. • Excellent clinical, immunological and

rest of body) and a dry cough. Considerable predominantly virological response to therapy.

• Examination: Afebrile, generalized lymphadenopathy, disseminated macrophage infiltrate with • Lost to follow up after 7 months when

papular-verrucous rash (some lesions were impetiginous), liver enlarged some poorly-formed CD4 116 cells/µl and VL LDL

clinically. granulomas.

• Laboratory: Liver function tests revealed a predominant cholestatic

pattern which slowly resolved on antifungal therapy.

• Radiology: normal chest radiograph, normal abdominal ultrasound

(normal but echogenic liver was reported).

2. 29-year-old • Background: HIV-infected; CD4+ T-lymphocyte count = 10 cells/µl; Positive (skin PAS stain-positive yeasts (3 • Amphotericin B for 14 days then

Black male previous diagnosis of pulmonary tuberculosis in 2003; commenced on biopsy) to 7 µm diameter) with D4T/3TC/EFV.

retreatment regimen for pulmonary tuberculosis in 2008 (diagnosis evidence of narrow-based • Good clinical, immunological and

based on symptoms [cough, night sweats] and a compatible chest budding present in virological response despite

radiograph) → no symptomatic improvement on anti-tuberculosis superficial dermis. Minimal inadvertently not receiving any

therapy. mixed perivascular maintenance antifungal therapy.

• History: Presented one month after initiation of TB treatment with a inflammatory infiltrate. • Latest follow up at 40 months, CD4

! 15! Case Demographic Background, clinical presentation, laboratory and radiology findings Culture of Histology of skin biopsy Treatment and outcome number information Emmonsia sp.

four-week history of fever and a two-week history of a diffuse non- 824 cells/µl, VL LDL.

itchy, papular rash.

• Examination: The papules were scattered over his whole body,

including his lips, his palms and soles. Some had central umbilication.

• Laboratory: Four sputum specimens submitted for tuberculosis

microscopy were auramine stain negative.

• Radiology: Hilar lymphadenopathy on chest radiograph; abdominal

ultrasound normal.

3. 56-year-old • Background: HIV-infected; CD4+ T-lymphocyte count = 5 cells/µl Positive (skin Dermis contained PAS • Respiratory failure responded well to

Black male • History: One-month history of profound loss of weight, fever and a biopsy) stain-positive yeasts (3 to 7 high-dose trimethoprim-

productive cough, including some hemoptysis over the previous week. . µm diameter), some with sulfamethoxazole for presumed

• Examination: Wasted, generalized lymphadenopathy, extensive narrow-based budding and pneumonia.

maculopapular rash, tachypnoeic with type one respiratory failure, liver minimal surrounding • He was commenced on amphotericin

and spleen were not enlarged. inflammation. B 1 mg/kg/day when the results of a

• Laboratory: Several blood, urine and sputum cultures were negative for skin biopsy revealed features of a deep

tuberculosis; two sputum cultures were positive for a non-tuberculous fungal infection. He was then switched

Mycobacterium species (not further speciated). to itraconazole due to creatinine

• Radiology: Chest radiograph revealed a dense opacification in the right increase to 314 µmol/L.

lower zone and subtle ground glass opacification in the rest of his • Initially responded to therapy – skin

fields. lesions and general condition

improved. Later patient became

confused, drowsy and refused oral

! 16! Case Demographic Background, clinical presentation, laboratory and radiology findings Culture of Histology of skin biopsy Treatment and outcome number information Emmonsia sp.

intake, followed by steady

deterioration in condition, despite the

introduction of empiric anti-

tuberculous therapy. He demised on

day 18 of antifungal therapy.

4. 38-year old • Background: HIV infection was diagnosed 6 years prior to presentation; Positive (skin Small PAS stain-positive • He was commenced on

White male CD4+ T-lymphocyte count = 118 cells/µl; no previous opportunistic biopsy) yeasts and minimal NVP/ZDV/3TC 6 weeks after starting

infections. Anti-tuberculosis treatment was initiated based on inflammation (3 to 7 µm TB therapy.

constitutional symptoms and a suggestive chest radiograph. diameter) were present in • Amphotericin B for 9 days

• History: He continued to lose weight and became confused on anti- the dermis. • Demised on day 9 of antifungal

tuberculosis treatment. Despite switching isoniazid to ofloxacin, treatment

encephalopathic features worsened.

• Examination: He had a generalized macular rash for 2 months

preceding initiation of antiretroviral treatment (ART), but these lesions

became larger and more erythematous on ART.

Laboratory: CSF was acellular with normal protein and glucose levels.

CSF fungal culture, CLAT and a PCR for JC Virus were negative.

Three sputum specimens and one CSF specimen submitted for culture

of M. tuberculosis were negative.

Radiology: Nodular opacifications were noted on his chest radiograph.

The nodular infiltrate became more prominent despite anti-tuberculosis

treatment. Numerous small lymph nodes in the celiac and para-aortic

! 17! Case Demographic Background, clinical presentation, laboratory and radiology findings Culture of Histology of skin biopsy Treatment and outcome number information Emmonsia sp.

region were noted on abdominal ultrasound. Asymmetrical cerebral

atrophy and non-enhancing, non-space demanding white matter lesions

were observed in occipital and cerebellar lobes on computed

tomography of the head (features compatible with progressive

multifocal leukoencephalopathy).

5. 34-year-old • Background: HIV-infected; CD4+ T-lymphocyte count = 13 cells/µl; Positive (blood Perivascular interstitial • Amphotericin B IV for 14 days

Black male no history of previous opportunistic infections. culture) inflammation with followed by itraconazole for one

• History: Six-week history of fever, skin lesions and mild respiratory lymphocytes, nuclear debris month. Then temporarily received 6

symptoms. and yeast cells. The PAS weeks of fluconazole due to the non-

• Examination: Pyrexial. purplish, papular skin lesions, some with with diastase stain availability of itraconazole.

central necrosis over his face, trunk and extremities. One seizure was confirmed numerous yeasts Itraconazole was then re-commenced.

recorded. (2 to 5 µm diameter) in the • Started on D4T/3TC/LPV/r.

• Laboratory: A dimorphic fungus grew from blood cultures (BACTEC upper dermis. • Excellent clinical, immunological and

MYCO/F-Lytic) after 156 hours. Standard blood cultures were virological response to therapy.

negative. A skin biopsy was only submitted for histology. CSF was • Latest follow up at 22 months – CD4

normal (negative CSF fungal culture and CLAT). Numerous cultures 426 cells/µl, VL LDL.

for M. tuberculosis were negative.

• Radiology: Non-specific bilateral nodular and peri-hilar infiltrates

with right peri-hilar lymphadenopathy were noted on chest radiograph;

a heterogenous mass (15 mm by 13 mm) was noted in the right kidney

(suggestive of papillary necrosis and possible tuberculosis) on

abdominal ultrasound; no intra-abdominal lymphadenopathy was

! 18! Case Demographic Background, clinical presentation, laboratory and radiology findings Culture of Histology of skin biopsy Treatment and outcome number information Emmonsia sp.

noted; computed tomography of the brain revealed no abnormalities.

6. 31-year-old • Background: HIV-infected; CD4+ T-lymphocyte count = 1 cell/µl; Positive (bone Numerous small PAS stain- • Amphotericin B for 12 days followed

Black female commenced on TDF/3TC/NVP and cotrimoxazole in 2010. Six marrow biopsy) positive yeasts in the by itraconazole.

months later, patient was started on treatment for tuberculosis based dermis. • 3TC/D4T/LPV/r commenced 7 days

on left pleuritic chest pain and right hilar lymphadenopathy on chest after completing amphotericin B

radiograph. A week later, she had a severe drug-induced hepatitis treatment.

which resolved on stopping her anti-tuberculosis treatment. • Patient had rapid resolution of skin

• History: Six weeks later, she developed an extensive macular papular lesions and improvement in general

rash with fever and loss of weight. condition when she was commenced on

• Laboratory: Enterococcus faecalis urinary tract infection; Parvovirus amphotericin B.

B19-related aplastic anemia (diagnosed on bone marrow, PCR and • Last to follow up after 4 months.

serology and treated with red cell transfusions). A skin biopsy

(January 2011) showed evidence of a deep fungal infection but was

not sent for fungal culture. She had evidence of virological and

immunological failure on ART - HIV-1 viral load (January 2011) =

105 RNA copies/ml (2 specimens); CD4+ T-lymphocyte counts were 2

cells/ìl and 5 cells/ìl.

• Radiology: Subtle nodular infiltrate on chest radiograph and mild

intrahepatic biliary dilatation and a small pericardial effusion noted on

abdominal ultrasound.

7. 40-year-old • Background: HIV-infected (admission diagnosis); CD4+ T-lymphocyte Positive for Provisional histology report • He was treated for secondary syphilis,

Black male count = 16 cells/µl; no previous opportunistic infections Emmonsia species suggested a diagnosis of commenced on anti-tuberculous therapy

! 19! Case Demographic Background, clinical presentation, laboratory and radiology findings Culture of Histology of skin biopsy Treatment and outcome number information Emmonsia sp.

• History: Three-week history of cough, fever, loss of weight and a diffuse based on Kaposi’s sarcoma. Final and discharged 16 days after admission.

papular rash on his face and trunk. phenotypic report concluded there was • The provisional histology report of his

• Examination: He was severely wasted, had generalized identification alone no evidence of Kaposi’s skin biopsy indicated Kaposi’s

lymphadenopathy and oral and . (skin biopsy) sarcoma. Dermis revealed sarcoma. He was discharged on no

• Laboratory: Pancytopenia, positive VDRL (titer 16), positive TPHA. very mild, mixed antifungal therapy.

Multiple cultures for tuberculosis were negative. inflammatory infiltrate and • He received 14 days of fluconazole 200

• Radiology: Patchy opacification in the left mid-zone was noted on chest small PAS stain-positive (3 mg QD as an inpatient for presumed

radiograph and a small volume of ascites (but no lymphadenopathy) and to 7 µm diameter) yeasts. esophageal candidiasis and noted an

a small pericardial effusion was noted on abdominal ultrasound. Mild perivascular improvement in his skin lesions and

inflammation. general condition during this time.

• Commenced on ART 3 weeks post-

discharge.

• Excellent clinical, immunological and

virologic response to treatment.

• Latest follow up at 36 months – CD4

308 cells/µl, VL LDL.

8. 33-year-old • Background: Diagnosed with HIV infection in 2004; CD4+ T- Positive (skin Small 3-7 µm PAS stain- • Commenced on D4T/3TC/EFV

Black female lymphocyte count = 60 cells/µl. Responded well to treatment for drug- biopsy) positive yeasts, some with • RHE converted to azithromycin and

susceptible M. tuberculosis infection in 2009. She had no response to narrow-based budding in ethambutol for presumed

streptomycin and RHZE was commenced 2010 for a syndrome of loss of dermis with moderate Mycobacterium avium intracellulare

weight, nausea, weakness and mild night sweats. amount of surrounding infection

• History: She was commenced on D4T/3TC/EFV 2 months after starting inflammation • Admitted for amphotericin B one

! 20! Case Demographic Background, clinical presentation, laboratory and radiology findings Culture of Histology of skin biopsy Treatment and outcome number information Emmonsia sp.

TB therapy and had a rapid increase in the number and size of papules month after starting ART.

all over her body which had been noted 2 weeks prior to the introduction Amphotericin B switched to

of ART. She also had worsening of nausea, weakness and developed itraconazole after one dose due to rapid

fever. deterioration in renal failure.

• Laboratory: Four sputum cultures were negative for M. tuberculosis. A • EFV was changed to boosted

blood culture at this time grew a non-tuberculous Mycobacterium which Lopinavir.

was not further speciated. At this time she also had a hemoglobin of 5 • At a follow-up visit in January 2011,

g/dL and creatinine of 166 µmol/L and a urine protein: creatinine ratio of skin lesions had almost completely

2.02 g/µmol. Blood culture (BACTEC MYCO/F-Lytic) positive for resolved, weight had increased by 4 kg

yeasts (some intracellular) at 5 days. and patient felt considerably better.

• Radiology: No abnormalities noted on chest radiograph. • Latest follow up at 21 months, all

symptoms resolved, CD4 319 cells/µl,

VL LDL.

9. 27-year-old • Background: HIV-infected; admitted 2011 with pneumonia and a skin Positive (blood PAS and Grocott’s stain- • Commenced on second-line TB

Black female rash. She had been on ART for 2 years and was failing therapy. Her cultures) positive fungi therapy

HIV-1 VL was 48 109 RNA copies/ml and her CD4+ T-lymphocyte • Switched to second-line ART

count dropped from 105 to 7 cells/µl. She had been fully treated for • Commenced amphotericin B 0.7

pulmonary tuberculosis in 2009. mg/kg for 2 weeks followed by

• History: She was febrile with a productive cough, bilateral wheezing on itraconazole 200 mg twice daily. She

chest auscultation and a skin rash comprising dark nodules over her completely recovered from transient

entire body. renal dysfunction probably related to

• Laboratory: Two separate sputum samples from admission were AFB- amphotericin B.

! 21! Case Demographic Background, clinical presentation, laboratory and radiology findings Culture of Histology of skin biopsy Treatment and outcome number information Emmonsia sp.

positive and cultured M. tuberculosis. A skin biopsy specimen was sent • Excellent clinical and virological

for histology but not for culture. Two blood cultures (BACTEC response to therapy.

MYCO/F-Lytic) were positive for fungal growth at 123 and 163 hours. • Latest follow up at 12 months, CD4

A standard aerobic blood culture (BACTEC) was positive for fungal 319 cells/µl, VL LDL.

growth at 110 hours.

• Radiology: Possible hilar adenopathy on chest radiograph and liver

showed increased echogenicity on abdominal ultrasound.

10. 41-year-old • Background: HIV-infected; CD4+ T-lymphocyte count = 32 cells/µl. Positive (skin Mixed nodular non- • Commenced on empiric TB therapy

Black female • History: Presented with fever, diarrhoea and loss of weight. biopsy) granulomatous inflammatio without improvement.

• Examination: Multiple pigmented purple papules over entire body, some n in the dermis. PAS and • Received 2 weeks of fluconazole 200

with necrotic centers. Grocott stains revealed scant mg QD for presumed esophageal

• Laboratory: Several sputum and blood cultures and a bone marrow y budding yeasts in the candidiasis from 15 January 2011.

biopsy and culture showed no evidence of M. tuberculosis infection. upper dermis. • Commenced ART on 15/02/11.

• Radiology: Bilateral nodular infiltrate on chest radiograph and • She lost 12.5 kg while on TB treatment

mediastinal lymphadenopathy and bilateral lung nodules with associated and her clinical condition only

ground glass opacification on chest computed tomography. improved after receiving fluconazole

and the subsequent ART.

• Good clinical, virological and

immunological response.

• Latest follow up at 14 months, CD4

229 cells/µl, VL LDL.

! 22! Case Demographic Background, clinical presentation, laboratory and radiology findings Culture of Histology of skin biopsy Treatment and outcome number information Emmonsia sp.

11. 38-year-old • Background: HIV-infected; CD4+ T-lymphocyte count = 49 cells/µl. Positive (skin Conspicuous inflammatory • Treated with amphotericin B for 2

Black male Initiated TDF/3TC/EFV and three months later treatment for culture- biopsy) infiltrates noted within the s weeks, followed by itraconazole 200mg

confirmed, multidrug-resistant pulmonary TB. uperficial dermis composed daily.

• History: Initially febrile and extremely weak (bed-bound). Fever settled predominantly of macropha • Excellent clinical, immunological and

and strength improved whilst receiving inpatient management for TB. ges and lymphocytes. Focal virological response. (CD4+ T-

Two months after commencing TB therapy, he developed hyper- micro-abscesses were noted. lymphocyte count = 142 cells/µl, VL

pigmented crusted papules and plaques on his right arm and over his Prominent small budding undetectable 6 months after starting

face. He also felt a return of the general weakness. fungal yeasts within the der ART).

• Laboratory: Latest sputum auramine stain and culture-negative for TB. mis were seen using • At latest follow up at 12 months, the

• Radiology: Nodular opacification of right lower zone on chest PAS and Grocott’s stains. patient was asymptomatic and his sputa

radiograph. were auramine stain and culture

negative for TB.

12. 34-year-old • Background: HIV-infected; CD4+ T-lymphocyte count = 32 cells/µl. Positive (skin Yeasts seen with Grocott’s • Good response to itraconazole and

Black male • History: One-month history of loss of weight, fever and skin lesions. On biopsy) stain; pronounced continuation of ART.

ART, loss of weight and fever persisted and skin lesions worsened granulomatous • After two months of antifungal therapy,

(increase in size and number of papules on face, trunk and limbs) and he inflammation. his skin lesions had almost completely

became bed bound due to profound weakness. Admitted 6 weeks into resolved, his weight and strength were

ART bedbound improved and he was walking

• Laboratory: Hemoglobin 4.5 g/dl (admission). normally.

• Radiology: Hilar lymphadenopathy on chest radiograph. • He decided to stop his itraconazole

after 3 months of therapy as he felt he

was back to normal. Eight months after

! 23! Case Demographic Background, clinical presentation, laboratory and radiology findings Culture of Histology of skin biopsy Treatment and outcome number information Emmonsia sp.

starting his antifungal therapy, he was

asymptomatic on ART. CD4+ T-

lymphocyte count increased to 285

cells/µl. VL not checked.

13. 27-year-old • Background: HIV-infected; two previous episodes of pulmonary TB Positive (blood Dense dermal lymphohistioc • Patient received no further antifungal

Mixed Race were fully treated. Commenced ART in 2008 with a good initial culture) ytic inflammatory infiltrate. therapy and died 2 days after

female response (CD4+ T-lymphocyte count increased to 513 cells/µl and VL Numerous yeasts, focally d admission.

15 was undetectable), but defaulted ART in 2009 due to metamphetamine emonstrating budding, were

abuse. Treated with 14 day course of fluconazole for presumed seen on PAS and Grocott’s s

oesophageal candidiasis and recommenced ART again in 2011. taining.

• History: At this point her body mass index was 11.7, she was

wheelchair-bound due to profound weakness and she was noted to have

a few dark crusting lesions on her trunk and left arm. She returned one

month later feeling somewhat improved (now able to walk with

assistance) but her skin lesions were now much larger and more

widespread. Admitted two weeks later complaining of fatigue, fevers

and weakness. Multiple raised, purple skin lesions were visible on her

face and chest.

• Laboratory: Two blood cultures were taken on the day of admission. She

received appropriate antibiotic therapy for an Escherichia coli

bacteremia. A fungus grew from blood culture after 2 days (identified as

Emmonsia species). Numerous yeasts were seen inside neutrophils on

! 24! Case Demographic Background, clinical presentation, laboratory and radiology findings Culture of Histology of skin biopsy Treatment and outcome number information Emmonsia sp.

the peripheral blood smear. Sputum specimens from August 2010 and

June 2011 were auramine and culture-negative for M. tuberculosis.

• Radiology: Bilateral diffuse patchy infiltrates on chest radiograph.

Abbreviations: DRUGS: AmpB Amphotericin B, AZT – Zidovudine, 3TC – Lamivudine, D4T – Stavudine, TDF – Tenofovir, NVP – Neviripine, EFV – Efavirenz, LPV/r – Boosted Lopinovir, RHZE – Rifampicin, Isoniazid, Pyrazinamide, Ethambutol. OTHERS: CXR – Chest X Ray, CLAT – Cryptococcal Latex Antigen Test, CSF – cerebrospinal fluid, F – Female, LAD – Lymphadenopathy, LDL – lower than detectable limit, M – Male, MAC – Mycobacterium Avium Intracellulare, MTB – mycobacterium tuberculosis, PML – Progressive Multifocal Leukoencephalopathy, TB Tuberculosis, USS – Ultrasound scan, VL – Viral Load.

! 25! Table S2: Minimum inhibitory concentrations of nine antifungal drugs for Emmonsia sp. clinical isolates (n=6) and the type strain, Emmonsia sp. NCPF 4164. Emmonsia sp. Antifungal drug NCPF 4164 Isolate 3 Isolate 4 Isolate 5 Isolate 11 Isolate 12 Isolate 13

Amphotericin B 0.125 0.25 0.25 0.12 0.25 1 1

Itraconazole <0.03 <0.015 <0.015 <0.015 <0.015 <0.015 0.015

Fluconazole <0.125 0.5 0.5 0.25 0.25 <0.12 2

Voriconazole <0.03 <0.008 <0.008 <0.008 <0.008 <0.008 0.015

Posaconazole <0.03 <0.008 0.008 0.008 <0.008 <0.008 0.008

Caspofungin 0.125 >8 2 8 0.06 8 0.5

Micafungin <0.03 >8 >8 8 0.03 8 0.12

Anidulafungin 0.03 4 2 4 0.03 2 2

Flucytosine 16 >64 >64 >64 0.06 >64 >64

Notes. Data are minimum inhibitory concentrations expressed as mg/L. NCPF – National Collection of Pathogenic Fungi, UK.

! 26! References 1. White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics.

In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR Protocols: a guide to methods and applications. San Diego, CA, USA:

Academic Press, Inc., 315–322.

2. Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast

Fourier transform. Nucleic Acids Research 30:3059-3066’

3. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0.

Molecular Biology and Evolution 24:1596-1599

4. Swofford DL (2002) PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). Version 4. Sunderland,

Massachusetts: Sinauer Associates.

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6. Clinical and Laboratory Standards Institute/National Committee for Clinical Laboratory Standards. Reference method for broth

dilution susceptibility testing of yeasts: approved standard, 3rd ed. 2008, Wayne, PA.

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