MYCOTIC INFECTIONS IN THEMIDDLE EAST

(Basidiobolus and Rhinocladiella)Ziauddin Khan, PhD

Professor of Medical MycologyDepartment of Microbiology

Faculty of MedicineKuwait University

Basidiobolus and Basidiobolomycosis

TAXONOMICAL CONSIDERATIONS Zygomycota underwent major taxonomic

changes in 2007. Hibbet and others proposed to eliminate

Zygomycota and the taxa conventionally placedin Zygomycota were distributed among thephylum Glomeromycota.

Mucorales and Entomophthorales, whichcontain zoopathogenic fungi, and 2 other ordersincluding Kickxellales and Zoopagales wereraised to the rank of subphyla:Mucoromycotina, Entomophthoromycotina,Kickxellomycotina and Zoopagomycotina.

Zygomycota underwent major taxonomicchanges in 2007.

Hibbet and others proposed to eliminateZygomycota and the taxa conventionally placedin Zygomycota were distributed among thephylum Glomeromycota.

Mucorales and Entomophthorales, whichcontain zoopathogenic fungi, and 2 other ordersincluding Kickxellales and Zoopagales wereraised to the rank of subphyla:Mucoromycotina, Entomophthoromycotina,Kickxellomycotina and Zoopagomycotina.

TAXONOMIC CLASSIFICATION

OLD

NEW

Kwon-Chung Clin Infect Dis. 2012;54:S8-S15

A proposed new classification schemesof the kingdom Fungi

Basidiobolomycetes Neozygitomycetes EntomophothoromycetesBasidiobolus spp. Neozygitis spp. Conidiobolus spp.

Humber, 2012

Phylum: EntomophthoromycotaClass: Basidiobolomycetes

Order: Basidiobolales

Phylogenetic classification of entomophthoroidfungi

Class: BasidiobolomycetesOrder: Basidiobolales

Family: BasidiobolaceaeGenus: Basidiobolus

Gryganskyi et al. Persoonia 2013; 30;94-105.

Maximum likelihood phylogeny of Basidiobolomycotina

(secondary conidiogenesis)

Basidiobolus and the still formally undescribed genera Schizangiella andDrechslerosporium (LSU, SSU, RPB2, mtSSU, ITS).

Gryganskyi et al. Persoonia 2013; 30;94-105

Life Cycle of

Adapted from Mendoza et al. 2015

Basidiobolus ranarum in intestinal tract of bats,reptiles and amphibians

Intestinal contents of 14 (7%) of 200 bats belonging toRhinopoma hardwickei hardwickei Gray ('the lesser rat-tailed bat'), an insectivorous species captured from Delhiarea.

Intestinal tract of 50% of trapped reptiles and amphibiansfrom Florida were found colonized with a Basidiobolusspecies.

Basidiobolus ranarum in intestinal tract of bats,reptiles and amphibians

Intestinal contents of 14 (7%) of 200 bats belonging toRhinopoma hardwickei hardwickei Gray ('the lesser rat-tailed bat'), an insectivorous species captured from Delhiarea.

Intestinal tract of 50% of trapped reptiles and amphibiansfrom Florida were found colonized with a Basidiobolusspecies.

Chaturvedi et al. Sabouraudia. 1984;22:185-9.

Okafor et al. Sabouraudia. 1984;22:47-51.

Epidemiology• Most cases of basidiobolomycosis have been

reported from tropical and subtropical regions ofAfrica, South America, and Asia

• It usually causes subcutaneous infections• Infections caused by B. ranarum are mainly

diagnosed in children (80% under the age of 20years) with a male/female ratio of 3:1.

• B. ranarum also causes chronic granulomatousinfection of the gastrointestinal tract in apparentlyimmunocompetent patients, mostly from Arizona(USA) and Middle East

• Most cases of basidiobolomycosis have beenreported from tropical and subtropical regions ofAfrica, South America, and Asia

• It usually causes subcutaneous infections• Infections caused by B. ranarum are mainly

diagnosed in children (80% under the age of 20years) with a male/female ratio of 3:1.

• B. ranarum also causes chronic granulomatousinfection of the gastrointestinal tract in apparentlyimmunocompetent patients, mostly from Arizona(USA) and Middle East

Basidiobolus is a ubiquitous fungus

The majority of cases have been reported in South America, Africa and tropicalAsia. However, recently this fungus has been classified as an emerging invasivefungal infection in desert regions of the US Southwest and Middle East causinggastrointestinal basidiobolomycosis.

Subcutaneous“Bathing suitdistribution”

Basidiobolomycosis: Clinical types

GastrointestinalColorectal

involvement

Complications: Small bowel, Liver, Gall bladder,Kidney, Pancreas, other intraabdominal organs

Clinical Manifestations:Basidiobolomycosis

The presenting feature is a single painless,unilateral, well-circumscribed subcutaneousmass that usually affects the buttock or thethigh but can also be seen in the arm, theneck, the face or the trunk.The disease starts as a single nodule that

progressively grows. The swelling is oftendescribed as woody and hard.

The presenting feature is a single painless,unilateral, well-circumscribed subcutaneousmass that usually affects the buttock or thethigh but can also be seen in the arm, theneck, the face or the trunk.The disease starts as a single nodule that

progressively grows. The swelling is oftendescribed as woody and hard.

The swelling over thigh in an 18-month-old female ofthree months duration caused by Basidiobolusranarum (culture and histopathology proven). Treatedwith KI (3 times a day) and lesion resolved within 1month

Naniwadekar et al. 2009A case from Karad, Karnataka

Buttock (toilet leaves?)Induration and multiple nodules in theperineum and labia (biopsy site seen)Radjou & Rajesh Ind J Microbiol. 2011; 29:186-188

A case fro Puducherry

HistopathologyChronic inflammatory process can be seen with

small abscesses surrounded by a granulomatoustissue reaction.A strong eosinophilic perihyphal reaction is often

observed (Splendore-Hoeppli phenomenon) that isvariable in size (2-6 µm).Board irregular hyphae (4-30µm) with thin walls and

rare septation can be seen, singly or in clusters.There is no invasion of blood vessels or infection of

tissue.

Chronic inflammatory process can be seen withsmall abscesses surrounded by a granulomatoustissue reaction.A strong eosinophilic perihyphal reaction is often

observed (Splendore-Hoeppli phenomenon) that isvariable in size (2-6 µm).Board irregular hyphae (4-30µm) with thin walls and

rare septation can be seen, singly or in clusters.There is no invasion of blood vessels or infection of

tissue.

Splendore-Hoeppli phenomenon (H &E) and Broad, aseptate hyphal fragments(GMS)

CULTUREBiopsy specimen should be cultured immediately

as B. ranarum dies quickly in tissues kept in arefrigerator.Tissue biopsy should be minced and not

homogenized. Homogenization in a tissue grindershould be avoided, because it decreases cultureyield by destroying hyphae.SDA with antibiotics can be used. Cycloheximide

should not be used in SDA. Incubation at 25°-30° C for 2-5 days.

Biopsy specimen should be cultured immediatelyas B. ranarum dies quickly in tissues kept in arefrigerator.Tissue biopsy should be minced and not

homogenized. Homogenization in a tissue grindershould be avoided, because it decreases cultureyield by destroying hyphae.SDA with antibiotics can be used. Cycloheximide

should not be used in SDA. Incubation at 25°-30° C for 2-5 days.

Gastrointestinal Basidiobolomycosis

CASE 1: A 30-year-old Bangladeshiwith rectal bleeding

• Rectal mass ~10 cm large polypoid mass onlower third of rectum

• Provisional diagnosis: Piles, rectal carcinoma• First biopsy: epithelioid cell granuloma

infiltrated with eosinophils- Crohn’s diseaseHistopathology: Fungal elements were missed

• Second biopsy: Histopathology: Fungalelements missed, culture yielded a fungus

• Rectal mass ~10 cm large polypoid mass onlower third of rectum

• Provisional diagnosis: Piles, rectal carcinoma• First biopsy: epithelioid cell granuloma

infiltrated with eosinophils- Crohn’s diseaseHistopathology: Fungal elements were missed

• Second biopsy: Histopathology: Fungalelements missed, culture yielded a fungus

Khan et al. Clin Infect Dis. 1998;26:521-3

B. ranarum colonies on SDA

Khan et al. Clin Infect Dis 1998;26:521

Zygospores

Immunodiffusion test

Case 2: A 41-year-old Indian male with historyof repair of a left inguinal hernia

• Examination: palpable, nodular mass near rt.hypochondrium,

• Ultrasound: thickening of ascending colon andcecum and renal involvement

• Provisional diagnosis: Intestinal tuberculosis

• Examination: palpable, nodular mass near rt.hypochondrium,

• Ultrasound: thickening of ascending colon andcecum and renal involvement

• Provisional diagnosis: Intestinal tuberculosis

Khan et al. J Clin Microbiol. 2001 ;39:2360-3

An open segment of large intestineshowing a thick-walled mucosa with acobblestone appearance mimickingCrohn's disease

Hematoxylin-and-eosin-stained sectionof intestinal mucosa showing a crosssection of a B. ranarum hypha (arrow)surrounded by the Splendore-Hoeppliphenomenon and many eosinophils

KOH-calcofluor white mount showing broad, non-septate, branched hyphae in urine sediment

The isolate was resistant to Amphotericin B(4 µg/ml) and itraconazole (8 µg/ml)

Serodiagnosis

Ab-I Ab-II

Ag

Ag-whole cell homoginate; patients’ seraAb-I and Ab-II

A case from Yemen/SA

Saeed et al. JMM CR 2014

A 12-year-old Yemeni boy, living in Saudi Arabia, was referred with a2-month history of diffuse abdominal pain, non-bilious vomiting,poor appetite and weight loss. percutaneous liver biopsy underultrasound guidance and sent for histopathological evaluation, whichrevealed granulomatous lesions rich in eosinophils with broadaseptate fungal hyphae surrounded by eosinophilic material(Splendore–Hoeppli phenomenon).

Abdominal CT showing multiple small, low-attenuation lesionsscattered throughout both lobes of the liver, some showing ringenhancement Saeed et al. JMM CR 2014

Liver CT after treatment with itraconazole treatment.Complete resolution after 12 months.

Saeed et al. JMM CR 2014

Gastrointestinal basidiobolomycosis accompanied by liverInvolvement

A case from Iran

Granuloma, Eosinophilic Infiltration andSplendore-Hoeppli Phenomenon (Arrow)

Ejtehadi et al. Iran Red Crescent Med J. 2014; 16: e14109.

Gross Pathology of theTerminal Ileum and RightColon Shows Ulceration in theCecum (Arrow)

A 41-year-old lady from Shiraz, south of Iran. CT-scan of the abdomen and pelvicshowed increased thickness of the cecum with infiltrative process and hypoattenuatinglesions in the liver, supporting liver abscesses or metastasis with central necrosisDiagnosis was based on histopathologic observation and response to itraconazoletherapy (200 mg twice a day for 4 months)

A 24-year-old woman from SaudiArabia with type I diabetes mellituspresented to the emergencydepartment with a 4-day history ofsevere abdominal pain, nausea,vomiting, abdominal distension, andconstipation. Magnetic resonanceimaging of the abdomen showed 2long segments of circumferentialheterogeneous enhancing soft tissuemasses involving the descendingcolon and descending sigmoid colonand multiple different-size hepaticlesions. Isolate identified by D1/D2sequencing. Treated withvoriconazole.

.

Gastrointestinal basidiobolomycosis with liver InvolvementA case from Saudi Arabia

A 24-year-old woman from SaudiArabia with type I diabetes mellituspresented to the emergencydepartment with a 4-day history ofsevere abdominal pain, nausea,vomiting, abdominal distension, andconstipation. Magnetic resonanceimaging of the abdomen showed 2long segments of circumferentialheterogeneous enhancing soft tissuemasses involving the descendingcolon and descending sigmoid colonand multiple different-size hepaticlesions. Isolate identified by D1/D2sequencing. Treated withvoriconazole.

Lactophenol cotton blue stain ofgrowth from fine needle aspirationmaterial from the liver lesion,X400

Alhuraiji et al. Clin Infect Dis. 2014; 58:990

Eosinophilic granulomatous gastrointestinal andhepatic abscesses attributable to basidiobolomycosis

in Iraqi Kurdistan (2009-2012)

- All males (6 cases)- Age: 17 months -59 years- Sites affected: Cecum and/or colon-5 cases, oropharyngeal and

transverse colon mass-1- Diagnosis- Histopathological (sparsely septate hyphae)- Treatment: oral itraconazole -5, all responded, became

asymptomatic (4-6 months);- One (17 months old) died after 2days of start of amphotericin B

Hassan et al. BMC Infect Dis. 2013; 13: 91.

- All males (6 cases)- Age: 17 months -59 years- Sites affected: Cecum and/or colon-5 cases, oropharyngeal and

transverse colon mass-1- Diagnosis- Histopathological (sparsely septate hyphae)- Treatment: oral itraconazole -5, all responded, became

asymptomatic (4-6 months);- One (17 months old) died after 2days of start of amphotericin B

Characteristics of 72 Patients With GastrointestinalBasidiobolomycosis

Variables ValueAge, years 13 months-81 years, ~85% in pediatric age group

Male sex 59 (82%)

Country of residenceUnited StatesSaudi ArabiaIranKuwaitIraq/KurdistanBrazilOthers

19, 17 from Arizona16, 14 in <12 years18, 11 <12 years

2, Adults (Indian, Bangladeshi)6, 2 in< 12 years47 (Nigeria, Italy, Netherlands, UK, Egypt, Thailand)

United StatesSaudi ArabiaIranKuwaitIraq/KurdistanBrazilOthers

19, 17 from Arizona16, 14 in <12 years18, 11 <12 years

2, Adults (Indian, Bangladeshi)6, 2 in< 12 years47 (Nigeria, Italy, Netherlands, UK, Egypt, Thailand)

Onset of GIB1964–19941995–2008

864 (86%)

n=42

(Geramizadeh et al. 2012; Vikram et al. 2012; Al-Asmi et al. 2013)

Antifungal susceptibilities ofConidiobolus and Basidiobolus

Species Geometric mean MICs (mg/L)(no. ofisolatestested)

Amphotericin B

Fluconazole

Flucytosine

Itraconazole

Ketoconazole

Miconazole

Conidiobolus spp.(9)

Conidiobolus spp.(9) 3.1 107.5 234.6 11.3 20.7 11.3Basidiobolus spp.(8)* 2.7 14.8 165.9 1.8 1.0 3.9

Guarro et al. J. Antimicrob. Chemother. 1999;44557-560

*Voriconazole and Posaconazole also exhibit good activity

Management• Therapeutic recommendations for the treatment of

basidiobolomycosis can be made only based on empiricobservations

• Potassium iodide, azoles, triazoles, terbinafine, and amphotericinB have been all used.

• In general, Basidiobolus spp. display lower MICs for triazoles thanConidiobolus spp.

• Potassium iodide has been used alone or in combinationwith itraconazole.

• In addition, surgical removal of accessible nodules andreconstructive surgery are usually required

• Therapeutic recommendations for the treatment ofbasidiobolomycosis can be made only based on empiricobservations

• Potassium iodide, azoles, triazoles, terbinafine, and amphotericinB have been all used.

• In general, Basidiobolus spp. display lower MICs for triazoles thanConidiobolus spp.

• Potassium iodide has been used alone or in combinationwith itraconazole.

• In addition, surgical removal of accessible nodules andreconstructive surgery are usually required

Conclusions

• Basidiobolomycosis is a disease of tropical andsub-tropical regions, however, GIB ispredominantly seen in arid regions.

• GIB is presumably acquired through ingestion• Surgical intervention combined with prolonged

antifungal therapy (itraconazole) is the preferredtreatment option

• Basidiobolomycosis is a disease of tropical andsub-tropical regions, however, GIB ispredominantly seen in arid regions.

• GIB is presumably acquired through ingestion• Surgical intervention combined with prolonged

antifungal therapy (itraconazole) is the preferredtreatment option

R. mackenziei (cerebral pheohyphomycosis)R. aquaspersa (Chromoblastomycosis)R. basitona (rare agents of skin infections)R. similis (rare agents of skin infections)

Colonies olivaceous to black, velvety; long, erect, brown,unbranched sympodial conidiophores; 1-celled pale ellipsoidalconidia borne on crowded denticles. Conidiophores usually poorlydifferentiated from the vegetative hyphae; conidial apparatus oftenloosely branched; exophiala-like budding cells usually present inculture.

Order - Chaetothyriales:Genus - Rhinocladiella

R. mackenziei (cerebral pheohyphomycosis)R. aquaspersa (Chromoblastomycosis)R. basitona (rare agents of skin infections)R. similis (rare agents of skin infections)

Colonies olivaceous to black, velvety; long, erect, brown,unbranched sympodial conidiophores; 1-celled pale ellipsoidalconidia borne on crowded denticles. Conidiophores usually poorlydifferentiated from the vegetative hyphae; conidial apparatus oftenloosely branched; exophiala-like budding cells usually present inculture.

Arzanlou et al. 2007

Phylogenetic and morphotaxonomic revision of Ramichloridium and alliedgenera (based on partial sequences of the 28S (LSU) rRNA gene and the ITS region (ITS1,5.8S rDNA and TS2).

First fatal cerebral phaeohyphomycosis due to Rhinocladiellamackenziei

A 54-year-old male, construction workerwith a 2-day-history of fever, frontalheadache, dizziness, and facio-brachialtonic-clonic seizures and left-sidedhemiparesis. The patient was treated withintravenous amphotericin B deoxycholate(0.5 mg/kg/day) combined with oralitraconazole (200 mg twice daily),nevertheless, his neurological functiondeteriorated rapidly and ultimately thepatient died due to respiratory failurelater two weeks.

A case from Iran

A. CT scan showed a discrete, large, irregular, peripheral ring-enhancing necrotic masslesion in the right fronto-parietal region with perifocal edema; B. MRI)revealed a large,supratentorial, intracranial, hyper dense, right fronto-temporal, space occupying lesionwith contrast enhancement components Didehdar et al. 2010 J Mycol Medicale 2014

A 54-year-old male, construction workerwith a 2-day-history of fever, frontalheadache, dizziness, and facio-brachialtonic-clonic seizures and left-sidedhemiparesis. The patient was treated withintravenous amphotericin B deoxycholate(0.5 mg/kg/day) combined with oralitraconazole (200 mg twice daily),nevertheless, his neurological functiondeteriorated rapidly and ultimately thepatient died due to respiratory failurelater two weeks.

A. KOH mount: Pigmented,septate, branch fungalhyphae

B. H & E stain: showed necrosiswith dense and diffuse mixedinflammatory infiltrates

Rhinocladiella mackenziei in Iran

A. KOH mount: Pigmented,septate, branch fungalhyphae

B. H & E stain: showed necrosiswith dense and diffuse mixedinflammatory infiltrates

R. mackenziei was identified based on the sequencing ofinternal transcribed spacer (ITS rDNA region) (KJ140287

Didehdar et al. 2010 J Mycol Medicale 2014

R. mackenziei in a Woman Native to AfghanistanAn 80-year-old Afghaniwoman with a history ofischemic heart diseaseand colon cancer, livingin France for 20 years,with no travel to MiddleEast or to her nativecountry. Initially treatedwith voriconazole ,followed by oralposaconazole (400 mgBID). Four months aftersurgery, the patient wasconscious, with a stableneurological status anddisappearance of Rttemporal lesion.Discharged onposaconazole but died 7months later.

(A) Postcontrast axial T1-weighted MR image showingthe rim-enhancing left temporal lesion with a centralhypointensity. (B) The low ADC suggests the diagnosis ofabscesses.

An 80-year-old Afghaniwoman with a history ofischemic heart diseaseand colon cancer, livingin France for 20 years,with no travel to MiddleEast or to her nativecountry. Initially treatedwith voriconazole ,followed by oralposaconazole (400 mgBID). Four months aftersurgery, the patient wasconscious, with a stableneurological status anddisappearance of Rttemporal lesion.Discharged onposaconazole but died 7months later.Cristini et al. J Clin Microbiol. 2010;48:3451

59-year-old Qatarifemale with carcinomaof the left breastshowed a cyst-likeenhancing lesion of theright posterior-parietaland occipital regionswith edema. Treatedwith AMB, VOR and POSbut died.

A case from Qatar

Taj –Aldeen et al Med Mycol. 2010;48:546-56

59-year-old Qatarifemale with carcinomaof the left breastshowed a cyst-likeenhancing lesion of theright posterior-parietaland occipital regionswith edema. Treatedwith AMB, VOR and POSbut died.

A case from Qatar

Invasion of fungal hyphae of R. mackenziei into the brain tissueshowing right angle-branched hyphae and moniliform (bead-like)hyphae.

Taj –Aldeen et al.Med Mycol. 2010;48:546-56

GMS PAS

Reports from Kuwait (Case 1)

Pondos et al. Neurosurgery 1999; 45:372

A 58-year-old Kuwaiti woman, with a history of chronic renal failure requiringhemodialysis, presented with a 3-day history of left frontal headache, blurry vision,dizziness, and right-sided clumsiness. CT demonstrated multiple, ring-enhancing,cerebral lesions in the deep left parieto-occipital region. Biopsy yielded dark caseousfluid, which demonstrated long, branching, septate hyphae. Cultures grew R.obovoideum. Despite treatment with a combination of amphotericin B anditraconazole, the patient died.

56-year-old Egyptianemployed as a masonsince 1994 in Kuwait,presented withheadache (1 monthduration) andprogressive loss ofvision. Abdominalultrasound revealed acirrhotic liver and brainCT scan, revealed alarge circular lesion(arrow)

Reports from Kuwait (Case 2)

Large circular lesions with ring enhancing lesion in left occipital lobe

56-year-old Egyptianemployed as a masonsince 1994 in Kuwait,presented withheadache (1 monthduration) andprogressive loss ofvision. Abdominalultrasound revealed acirrhotic liver and brainCT scan, revealed alarge circular lesion(arrow)

Brain biopsy culture onSDA

KOH preparation of brainbiopsy

Slide culture preparation of R.mackenziei showingsympodially proliferating conidiogenous cells withmostly two conidia

Isolate resistant to Amphotericin B

Cerebral pheohyphomycosis due toR.mackenziei in Pakistan

Six cases: 5 M (30-75 years), 1 F (20 years),Residence: 3 Balochistan, 2 Sindh, I NotknownIdentification confirmed by DNA sequencingSurgical intervention and antifungal therapyComorbidities: Present in 4 patientsOutcome: 3 died, 2 survived, 1 lost for follow-up

Six cases: 5 M (30-75 years), 1 F (20 years),Residence: 3 Balochistan, 2 Sindh, I NotknownIdentification confirmed by DNA sequencingSurgical intervention and antifungal therapyComorbidities: Present in 4 patientsOutcome: 3 died, 2 survived, 1 lost for follow-up

Jabeen et al. CID 2011;52:213

2 cases

2 casesIND, AFG

Cerebral Phaeohyphomycosis due toRhinocladiella mackenziei

Predominantly a disease of the Middle EastKUWAIT

13 cases

6 cases

5 cases 46%

Saudi ArabiaQ, O, UAE,ISR, Moracco

28 cases

Pakistan

8 antifungal drugs against 10 clinicalisolates by CLSI microdilution method

Antifungal drugs against Rhinocladiellamackenziei

Rhinocladiella mackenziei gave MIC90 values (mg/L) forAmB, FLU, ITC, VOR, POS, ISA, CAS and ANI of 16, 64,0.25, 2, 0.063, 1, 8 and 8 mg/L, respectively. In vitroactivities have shown that all strains of Rhinocladiellamackenziei are resistance to AmB and echinocandindrugs. In contrast, POS, ITC, and ISA were the most activedrugs with high in vitro activity against R. mackenziei alsoconfirmed by animal experiments.

Rhinocladiella mackenziei gave MIC90 values (mg/L) forAmB, FLU, ITC, VOR, POS, ISA, CAS and ANI of 16, 64,0.25, 2, 0.063, 1, 8 and 8 mg/L, respectively. In vitroactivities have shown that all strains of Rhinocladiellamackenziei are resistance to AmB and echinocandindrugs. In contrast, POS, ITC, and ISA were the most activedrugs with high in vitro activity against R. mackenziei alsoconfirmed by animal experiments.

Badali et al. J. Atimicrobial Chemother. 2010

Thank youThank youfor yourfor yourkind attention…kind attention…

Fungi spare nobody

Thank youThank youfor yourfor yourkind attention…kind attention…

An ant infected withascomycetous fungus of the genus

Cordyceps