MYCOTIC INFECTIONS IN THE MIDDLE EAST (Basidiobolus and Rhinocladiella)

Ziauddin Khan, PhD Professor of Medical Department of Microbiology Faculty of Medicine Kuwait University Basidiobolus and TAXONOMICAL CONSIDERATIONS  underwent major taxonomic changes in 2007.  Hibbet and others proposed to eliminate Zygomycota and the taxa conventionally placed in Zygomycota were distributed among the phylum .  and , which contain zoopathogenic fungi, and 2 other orders including Kickxellales and were raised to the rank of subphyla: , Entomophthoromycotina, and . TAXONOMIC CLASSIFICATION

OLD

NEW

Kwon-Chung Clin Infect Dis. 2012;54:S8-S15 A proposed new classification schemes of the Fungi

Basidiobolomycetes Neozygitomycetes Entomophothoromycetes Basidiobolus spp. Neozygitis spp. Conidiobolus spp.

Humber, 2012 Phylogenetic classification of entomophthoroid fungi

Phylum: Class: Order: Basidiobolales Family: Genus: Basidiobolus

Gryganskyi et al. Persoonia 2013; 30;94-105. Maximum likelihood phylogeny of Basidiobolomycotina

(secondary conidiogenesis)

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

Gryganskyi et al. Persoonia 2013; 30;94-105 Life Cycle of

Adapted from Mendoza et al. 2015 in intestinal tract of bats, reptiles and amphibians

Intestinal contents of 14 (7%) of 200 bats belonging to Rhinopoma hardwickei hardwickei Gray ('the lesser rat- tailed bat'), an insectivorous species captured from Delhi area. Chaturvedi et al. Sabouraudia. 1984;22:185-9.

Intestinal tract of 50% of trapped reptiles and amphibians from Florida were found colonized with a Basidiobolus species. Okafor et al. Sabouraudia. 1984;22:47-51. Epidemiology • Most cases of basidiobolomycosis have been reported from tropical and subtropical regions of Africa, South America, and Asia • It usually causes subcutaneous infections • Infections caused by B. ranarum are mainly diagnosed in children (80% under the age of 20 years) with a male/female ratio of 3:1. • B. ranarum also causes chronic granulomatous infection of the in apparently immunocompetent patients, mostly from Arizona (USA) and Middle East Basidiobolus is a ubiquitous

The majority of cases have been reported in South America, Africa and tropical Asia. However, recently this fungus has been classified as an emerging invasive fungal infection in desert regions of the US Southwest and Middle East causing gastrointestinal basidiobolomycosis. Basidiobolomycosis: Clinical types

Subcutaneous “Bathing suit distribution”

Gastrointestinal Colorectal 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 subcutaneous mass that usually affects the buttock or the thigh but can also be seen in the arm, the neck, the face or the trunk. The disease starts as a single nodule that progressively grows. The swelling is often described as woody and hard. The swelling over thigh in an 18-month-old female of three months duration caused by Basidiobolus ranarum (culture and histopathology proven). Treated with KI (3 times a day) and lesion resolved within 1 month

Naniwadekar et al. 2009 A case from Karad, Karnataka Induration and multiple nodules in the Buttock (toilet leaves?) perineum and labia (biopsy site seen) Radjou & Rajesh Ind J Microbiol. 2011; 29:186-188 A case fro Puducherry Histopathology Chronic inflammatory process can be seen with small abscesses surrounded by a granulomatous tissue reaction. A strong eosinophilic perihyphal reaction is often observed (Splendore-Hoeppli phenomenon) that is variable 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) CULTURE

Biopsy specimen should be cultured immediately as B. ranarum dies quickly in tissues kept in a refrigerator. Tissue biopsy should be minced and not homogenized. Homogenization in a tissue grinder should be avoided, because it decreases culture yield 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 Bangladeshi with rectal bleeding

• Rectal mass ~10 cm large polypoid mass on lower third of rectum • Provisional diagnosis: Piles, rectal carcinoma • First biopsy: epithelioid cell granuloma infiltrated with eosinophils- Crohn’s disease Histopathology: Fungal elements were missed • Second biopsy: Histopathology: Fungal elements 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 history of repair of a left inguinal hernia

• Examination: palpable, nodular mass near rt. hypochondrium, • Ultrasound: thickening of ascending colon and cecum and renal involvement • Provisional diagnosis: Intestinal tuberculosis

Khan et al. J Clin Microbiol. 2001 ;39:2360-3 Hematoxylin-and-eosin-stained section An open segment of large intestine of intestinal mucosa showing a cross showing a thick-walled mucosa with a section of a B. ranarum (arrow) cobblestone appearance mimicking surrounded by the Splendore-Hoeppli Crohn's disease phenomenon and many eosinophils KOH-calcofluor white mount showing broad, non- septate, branched hyphae in urine sediment

The isolate was resistant to (4 µg/ml) and (8 µg/ml) Serodiagnosis

Ag

Ab-I Ab-II

Ag-whole cell homoginate; patients’ sera Ab-I and Ab-II A case from Yemen/SA

A 12-year-old Yemeni boy, living in Saudi Arabia, was referred with a 2-month history of diffuse abdominal pain, non-bilious vomiting, poor appetite and weight loss. percutaneous liver biopsy under ultrasound guidance and sent for histopathological evaluation, which revealed granulomatous lesions rich in eosinophils with broad aseptate fungal hyphae surrounded by eosinophilic material (Splendore–Hoeppli phenomenon). Saeed et al. JMM CR 2014 Abdominal CT showing multiple small, low-attenuation lesions scattered throughout both lobes of the liver, some showing ring enhancement Saeed et al. JMM CR 2014 Liver CT after treatment with itraconazole treatment. Complete resolution after 12 months.

Saeed et al. JMM CR 2014 A case from Iran Gastrointestinal basidiobolomycosis accompanied by liver Involvement

Gross Pathology of the Granuloma, Eosinophilic Infiltration and Terminal Ileum and Right Splendore-Hoeppli Phenomenon (Arrow) Colon Shows Ulceration in the Cecum (Arrow) A 41-year-old lady from Shiraz, south of Iran. CT-scan of the abdomen and pelvic showed increased thickness of the cecum with infiltrative process and hypoattenuating lesions in the liver, supporting liver abscesses or metastasis with central necrosis Diagnosis was based on histopathologic observation and response to itraconazole therapy (200 mg twice a day for 4 months)

Ejtehadi et al. Iran Red Crescent Med J. 2014; 16: e14109. . A case from Saudi Arabia Gastrointestinal basidiobolomycosis with liver Involvement

A 24-year-old woman from Saudi Arabia with type I diabetes mellitus presented to the emergency department with a 4-day history of severe abdominal pain, nausea, vomiting, abdominal distension, and constipation. Magnetic resonance imaging of the abdomen showed 2 long segments of circumferential heterogeneous enhancing soft tissue masses involving the descending colon and descending sigmoid colon Lactophenol cotton blue stain of and multiple different-size hepatic growth from fine needle aspiration lesions. Isolate identified by D1/D2 material from the liver lesion, X400 sequencing. Treated with voriconazole.

Alhuraiji et al. Clin Infect Dis. 2014; 58:990 Eosinophilic granulomatous gastrointestinal and hepatic 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. Characteristics of 72 Patients With Gastrointestinal Basidiobolomycosis Variables Value Age, years 13 months-81 years, ~85% in pediatric age group

Male sex 59 (82%) Country of residence United States 19, 17 from Arizona Saudi Arabia 16, 14 in <12 years Iran 18, 11 <12 years n=42 Kuwait 2, Adults (Indian, Bangladeshi) Iraq/Kurdistan 6, 2 in< 12 years Brazil 4 Others 7 (Nigeria, Italy, Netherlands, UK, Egypt, Thailand) Onset of GIB 1964–1994 8 1995–2008 64 (86%)

(Geramizadeh et al. 2012; Vikram et al. 2012; Al-Asmi et al. 2013) Antifungal susceptibilities of Conidiobolus and Basidiobolus

Species Geometric mean MICs (mg/L) (no. of isolates Amphot Fluconaz Flucytosi Itracona Ketocon Miconaz tested) ericin B ole ne zole azole ole Conidiob olus spp. (9) 3.1 107.5 234.6 11.3 20.7 11.3 Basidiob olus spp. (8)* 2.7 14.8 165.9 1.8 1.0 3.9 *Voriconazole and Posaconazole also exhibit good activity

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

• Therapeutic recommendations for the treatment of basidiobolomycosis can be made only based on empiric observations • Potassium iodide, azoles, triazoles, terbinafine, and amphotericin B have been all used. • In general, Basidiobolus spp. display lower MICs for triazoles than Conidiobolus spp. • Potassium iodide has been used alone or in combination with itraconazole. • In addition, surgical removal of accessible nodules and reconstructive surgery are usually required Conclusions

• Basidiobolomycosis is a disease of tropical and sub-tropical regions, however, GIB is predominantly seen in arid regions. • GIB is presumably acquired through ingestion • Surgical intervention combined with prolonged antifungal therapy (itraconazole) is the preferred treatment option Order - Chaetothyriales: Genus - Rhinocladiella R. mackenziei (cerebral pheohyphomycosis) R. aquaspersa () 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 ellipsoidal conidia borne on crowded denticles. Conidiophores usually poorly differentiated from the vegetative hyphae; conidial apparatus often loosely branched; exophiala-like budding cells usually present in culture. Phylogenetic and morphotaxonomic revision of Ramichloridium and allied genera (based on partial sequences of the 28S (LSU) rRNA gene and the ITS region (ITS1, 5.8S rDNA and TS2). Arzanlou et al. 2007 A case from Iran First fatal cerebral phaeohyphomycosis due to Rhinocladiella mackenziei

A 54-year-old male, construction worker with a 2-day-history of fever, frontal headache, dizziness, and facio-brachial tonic-clonic seizures and left-sided hemiparesis. The patient was treated with intravenous amphotericin B deoxycholate (0.5 mg/kg/day) combined with oral itraconazole (200 mg twice daily), nevertheless, his neurological function deteriorated rapidly and ultimately the patient died due to respiratory failure later two weeks. A. CT scan showed a discrete, large, irregular, peripheral ring-enhancing necrotic mass lesion in the right fronto-parietal region with perifocal edema; B. MRI)revealed a large, supratentorial, intracranial, hyper dense, right fronto-temporal, space occupying lesion with contrast enhancement components Didehdar et al. 2010 J Mycol Medicale 2014 Rhinocladiella mackenziei in Iran

A. KOH mount: Pigmented, B. H & E stain: showed necrosis septate, branch fungal with dense and diffuse mixed hyphae inflammatory infiltrates

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

Didehdar et al. 2010 J Mycol Medicale 2014 R. mackenziei in a Woman Native to Afghanistan An 80-year-old Afghani woman with a history of ischemic heart disease and colon cancer, living in France for 20 years, with no travel to Middle East or to her native country. Initially treated with voriconazole , followed by oral posaconazole (400 mg BID). Four months after surgery, the patient was (A) Postcontrast axial T1-weighted MR image showing conscious, with a stable the rim-enhancing left temporal lesion with a central neurological status and hypointensity. (B) The low ADC suggests the diagnosis of disappearance of Rt abscesses. temporal lesion. Discharged on posaconazole but died 7 Cristini et al. J Clin Microbiol. 2010;48:3451 months later. A case from Qatar 59-year-old Qatari female with carcinoma of the left breast showed a cyst-like enhancing lesion of the right posterior-parietal and occipital regions with edema. Treated with AMB, VOR and POS but died.

Taj –Aldeen et al Med Mycol. 2010;48:546-56 A case from Qatar

GMSInvasion of fungal hyphae of R. mackenzieiPAS into the brain tissue showing right angle-branched hyphae and moniliform (bead-like) hyphae.

Taj –Aldeen et al.Med Mycol. 2010;48:546-56 Reports from Kuwait (Case 1)

A 58-year-old Kuwaiti woman, with a history of chronic renal failure requiring hemodialysis, 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 caseous fluid, which demonstrated long, branching, septate hyphae. Cultures grew R. obovoideum. Despite treatment with a combination of amphotericin B and itraconazole, the patient died.

Pondos et al. Neurosurgery 1999; 45:372 Reports from Kuwait (Case 2) 56-year-old Egyptian employed as a mason since 1994 in Kuwait, presented with headache (1 month duration) and progressive loss of vision. Abdominal ultrasound revealed a cirrhotic liver and brain CT scan, revealed a large circular lesion (arrow)

Large circular lesions with ring enhancing lesion in left occipital lobe Brain biopsy culture on KOH preparation of brain SDA biopsy Slide culture preparation of R. mackenziei showing sympodially proliferating conidiogenous cells with mostly two conidia Isolate resistant to Amphotericin B Cerebral pheohyphomycosis due to R. mackenziei in Pakistan

Six cases: 5 M (30-75 years), 1 F (20 years), Residence: 3 Balochistan, 2 Sindh, I Not known Identification confirmed by DNA sequencing Surgical intervention and antifungal therapy Comorbidities: Present in 4 patients Outcome: 3 died, 2 survived, 1 lost for follow- up

Jabeen et al. CID 2011;52:213 Cerebral Phaeohyphomycosis due to Rhinocladiella mackenziei Predominantly a disease of the Middle East

KUWAIT

IND, AFG 2 cases 2 cases

Q, O, UAE, Saudi Arabia 13 cases ISR, Moracco 5 cases 46%

Pakistan 6 cases 28 cases Antifungal drugs against Rhinocladiella mackenziei 8 antifungal drugs against 10 clinical isolates by CLSI microdilution method

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

Badali et al. J. Atimicrobial Chemother. 2010 Fungi spare nobody Thank you for your kind attention…

An ant infected with ascomycetous fungus of the genus Cordyceps