Mucormycological Pearls

© by author Jagdish Chander GovernmentESCMID Online Medical Lecture College Library Hospital Sector 32, Chandigarh Introduction • Mucormycosis is a rapidly destructive necrotizing infection usually seen in diabetics and also in patients with other types of immunocompromised background • It occurs occurs due to disruption of normal protective barrier

• Local risk factors for mucormycosis include trauma, burns, surgery, surgical splints, arterial lines, injection sites, biopsy sites, tattoos and insect or spider bites

• Systemic risk factors for mucormycosis are hyperglycemia, ketoacidosis, malignancy,© byleucopenia authorand immunosuppressive therapy, however, infections in immunocompetent host is well described ESCMID Online Lecture Library • Mucormycetes are upcoming as emerging agents leading to fatal consequences, if not timely detected. Clinical Types of Mucormycosis

• Rhino-orbito-cerebral (44-49%) • Cutaneous (10-16%) • Pulmonary (10-11%), • Disseminated (6-12%) • Gastrointestinal© by (2 -author11%) • Isolated Renal mucormycosis (Case ESCMIDReports About Online 40) Lecture Library Broad Categories of Mucormycetes

Phylum: Glomeromycota (Former Zygomycota) Subphylum: Mucormycotina Mucormycetes Mucorales: Mucormycosis Acute angioinvasive infection in immunocompromised© by author individuals Entomophthorales: Entomophthoromycosis ESCMIDChronic subcutaneous Online Lecture infections Library in immunocompetent patients Agents of Mucormycosis Mucorales : Mucormycosis •Rhizopus arrhizus •Rhizopus microsporus var. rhizopodiformis •Lichtheimia (Mycocladus corymbiferus, Absidia corymbifera) •Rhizomucor pusillus •Mucor species •Mortierella species •Cunninghamella species •Apophysomyces elegans, A. variabilis, A. trapeziformis •Saksenaea vasiformis, ©S. erythrospora,by author S. oblongispora •Cokeromyces recurvatus and others species

Entomophthorales:ESCMID Online Entomophthoromycosis Lecture Library •Conidiobolomycosis – Conidiobolus coronatus •Basidiobolomycosis – Basidiobolus ranarum Apophysomyces spp.

© by author ESCMID Online Lecture Library Introduction • The mucoralean fungus Apophysomyces elegans is a thermotolerant species causing severe infections in humans, being the most common site of disease manifestation the cutaneous and subcutaneous tissues, with local invasion into muscle and fat tissue resulting in necrotizing fasciitis. • In contrast to the other fungi causing mucormycosis, which have a world-wide distribution and occur only rarely in immunocompetent hosts, A. elegans has been mainly reported in warm climate areas as© anbyemerging authorpathogen causing mostly cutaneous infections after injury to the cutaneous barrier. • This fungus was discovered in 1979 from Indian soils and until recentlyESCMIDit was considered Onlinethe Lectureonly species Libraryof the genus . Now, there are four species i.e. A .elegans, A. trapeziformis, A. ossiformis and A. variabilis. Patients & Methods • A total of 18 cases of primary cutaneous infection caused by the novel species Apophysomyces variabilis are being described. • The identification of the isolates was confirmed by ITS sequencing analysis. The clinical disease was characterized by an aggressive infection following traumatic implantation of the fungus, which caused a prompt tissue invasion eventually death of the patients. • The 18 isolates tentatively identified as A. elegans were sent to the mycology reference laboratory in Spain for molecular confirmation. • The ITS region of such isolates was sequenced and compared with those of the type strains©of thebyspecies authorof Apophysomyces . • These isolates were confirmed as Apophysomyces variabilis on the basisESCMIDof morphological Onlinefeatures Lectureof spored and molecularLibrarytyping . Patients and Methods  Patients: All patients diagnosed with cutaneous mucormycosis due to Apophysomyces species at Government Medical College Hospital (GMCH), Chandigarh, India, during one year period (2005 to 2014)  Clinical diagnosis was confirmed by KOH, Calcofluor wet mounts and histopathological examination of tissue specimens of infected site  Fungal culture was done on Sabouraud Dextrose Agar (SDA) incubated at 22◦C and 37◦C over a period of 4 weeks  Identification of fungi: Gross morphological examination along with microscopic examination using Lactophenol Cotton Blue (LCB) mount, particularly the spore morphology of the Mucormycete isolates  Molecular analysis: All the isolates sent to the Universitat Rovira i Virgili (Reus, Spain) for molecular analysis by sequencing internal transcribed spacer region and comparing with those of type strains of Apophysomyces species © by author  Antifungal susceptibility testing was done against these fungal isolates by using standard protocol  Clinical history: Detailed clinical history of these patients pertaining to ESCMIDrisk factors, immunocompromised Online status,Lectureetc., was taken Library  Outcome: Local debridement with liposomal amphotericin B (topical as well as I/V) was given and regular follow up of all patients was done Table 1. Clinical profile of patients infected by Apophysomyces spp. presented at GMCH, Chandigarh

No. Dates Name / Age / Sex / Residence Anatomical sites involved Clinical presentations Underlying illnesses Risk factors Isolates Outcome

1. 03.05.05 Roshni Devi, 32 F Anterior abdominal wall (left Fever, black discoloration 15×15cms on ant. abdominal wall with None Massage on abdomen for abdominal A. variabilis Expired after 7 days side) surrounding edema and induration pain 10 days prior

2. 22.08.05 Jay Kumar, 43 M Anterior abdominal wall (right 15×15cms debrided wound on right ant. abdominal wall, necrotic fat None H/o of injection on right ant. A. variabilis Recovered in 6 weeks side) at margin present, laterally skin discolored abdominal wall which developed period into small blister

3. 02.06.07 Balbir Singh, 29 M, Kapurthla (Punjab) Right arm, axilla and right Necrosed area on right arm at the site of Plaster of Paris cast None Plaster of Paris cast applied at the A. variabilis Recovered after 2 weeks part of chest wall extending to axilla and right part of chest wall site for fracture of right humerus

4. 13.08.07 Paramjeet Kaur, 35 F, Sangrur (Punjab) Left thigh & anterior Massive wound on left thigh & anterior abdominal wall with black Diabetes mellitus I.M. injection over left gluteal A. variabilis Expired after 1 week abdominal wall necrotic areas region 7 days prior

5. 22.04.09 Kanta Devi, 43 F, Dera Bassi (Punjab) Right sided chest wall Massive wound on right sided chest wall including the breast with Diabetes mellitus I/M injection over left gluteal region A. variabilis Expired after 6 days including breast black necrotic areas 7 days prior

6. 22.09.10 Bimla Devi, 50 F, Karnal (Haryana) Left gluteal area Fever, painful ulcerated lesion (16x20cm) on left gluteal region None I/M injection on left gluteal region A. variabilis Died two weeks later 15 days earlier

7. 11.10.10 Bimal Singh, 45 M, Mujaffarnagar Right gluteal and postero- Fever, necrosed area (30x24cm) on right gluteal region spreading None I/M injections on right gluteal A. variabilis Discharged after 15 days (Uttar Pradesh) lateral aspect of thigh posterolateral aspect of thigh region following appendectomy one month earlier

8. 15.10.10 Amarjeet Singh, 42 M, Mohali Right scapular region Painful ulcerated lesion (20x15cm) over right scapular area None No obvious risk factor could be A. variabilis Discharged after 15 days (Punjab) elucidated

9. 23.10.10 Kashmir Singh, 32 M, Chandigarh Swelling of right side of cheek He developed black necrotic patch on right side of hard palate. None No obvious risk factor could be A. variabilis Discharged after 15 days following tooth extraction, 2 elucidated weeks ago.

10. 06.11.10 Baljinder Kaur, 39 F, Ropar (Punjab) Right gluteal area Fever, painful ulcerated lesion (40x40cm) on right gluteal region Diabetes mellitus I/M injection on right gluteal region A. variabilis Left against medical one month earlier advice, presumed to have died

11. 29.04.11 Mayawati, 40 F, Bhiwani (Haryana) Left gluteal area Large ulcerative lesion (22x17mm) on left gluteal region None I/M injection on left gluteal region A. variabilis Left against medical one and a half month earlier. advice, presumed to have died

12. 25.06.11 Malkit Kaur, 52 F, Mohali (Punjab) Left gluteal region Painful, Large necrotic lesion (16x20cm) on left gluteal region Diabetes mellitus I/M injection on left gluteal region A. elegans Died after 10 days © by author one month earlier 13. 27.08.11 Amrt Lal, 50 M, Mauli Jagran, Left arm region Large necrotic area on left arm None Fall in bathroom 15 days earlier A. variabilis Discharged after 40 days Chandigarh

14. 07.08.13 Roopmati, 40 F, Pehoa (Haryana) Right gluteal area Necrotizing ulcerative area over right gluteal area None I/M injection on right gluteal region A. variabilis Expired after 1 week of 2 weeks earlier admission

15. 03.09.13 Jasveer Kaur , 22 F, Mohali (Punjab) Right gluteal area Large ulcerative lesion (25x20 mm) on right gluteal region None I/M injection on right gluteal region A. variabilis Expired after 1 month of 3 weeks earlier admission

16. 01.11.13 Kuldeep Singh, 40 M, (Punjab) Right Arm Necrotizing ulcerative area on right arm Diabetes mellitus Ulcerative lesion after motorcycle A. variabilis Discharged after 1 ESCMID Online Lecture Libraryaccident month of admission

17. 02.12.13 Mangat Rai, 59 M, Ambala (Haryana) Back and medial aspect of Necrotizing ulcerative lesion over backside and medial aspect of Diabetes mellitus Application of Dhatura on the local A. variabilis Expired after 2 days of right thigh right thigh wound admission

18. 07.09.14 Bhajan Kaur, 65 F, Barwala, Haryana Anterior abdominal wall A large necrotic patch over the anterior abdominal wall with None Massage on abdomen for abdominal A. variabilis Expired after 5 days. surrounding erythema pain 7-8 days prior KOH Wet Mount – Non-Septate Hyphae

Histopathology LCB © by author mount ESCMID Online Lecture Library Culture – Apophysomyces variablilis

© by author ESCMID Online Lecture Library Results • Total number of patients presenting with cutaneous mucormycosis due to Apophysomyces spp. over 10 years period was 18 as identified morphologically after 72 hours of incubation, subsequently confirmed by molecular typing

• There were 16 strains of A. variabilis and 1 strain of A. elegans, 1 is sent for sequencing

• Only 9 patients could be saved, 7 expired and 2 left against medical advice

• Two-third had H/o I/M injections and One-third had DM © by author • Antifungal suceptibility tsting was done againt amphotericin B (MIC ranges from 1-4), itraconazole (MIC ranges from 0.25-4), posaconazole (MIC ranges from 0.05-2), voriconazole (MIC ranges from 8-16), ESCMIDechinocandins i.e. Onlinecaspofungin, micafungin,Lectureanidulafungin Library(MIC ragnes from 16-32) Phylum : Glomeromycota (Former Zygomycota) Subphylum: Mucormycotina - Mucormycetes Sr. Family Genus Species No. 1. Mucoraceae Actinomucor, Actino. elegans, R. Mucor, Rhizopus, arrhizus, Mucor Rhizomucor racemosus, R. pusillus 2. Cunninghamellaceae Cunninghamella C. bertholletiae

3. Saksenaeaceae Apophysomyces, A. elegans complex, Saksenaea S.vasiformis complex 4. Syncephalastraceae ©Syncephalastrum by authorS. racemosum 5. Thamnidiaceae Cokeromyces C. recurvatus

6. ESCMIDLichtheimiaceae OnlineLichtheimia LectureL. corymbifera Library (Mycocladus or Absidia) Unusual Mucormycetes (Gomes et al, CMR; Vol. 24: 2011)

Sr. No. Genus/Species No. of Patients 1. Apophysomyces elegans 74 complex 2. Cunninghamella bertholletiae 43 3. Saksenaea vasiformis complex 42 4. Rhizomucor pusillus 22 5. Syncephalastrum racemosum 4 6. Cokeromyces©recurvatus by author 3 ESCMID7. Actinomucor Onlineelegans Lecture Library1 © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Timeline-Apophysomyces • 1979 – Misra et al – Discovered • 1982 – Ellis & Ajello – 1st Human Case USA • Subsequent Case Reports - USA (Texas, Arizona, Florida, Mississippi, Oklahoma, Georgia, Minnesota, Alabama, South Carolina), Venezuela, Colombia, Mexico, Australia, Saudi Arabia, Kuwait and The Netherlands. © by author • 2004 - Sri Lanka, Thailand - tsunami disaster • 2011ESCMID- Gomes Onlineet al – Lecture74 cases Library • 2012 - dela Cruz WP, Apophysomyces variabilis Indian Scenario

• 1993 - Lakshmi Hyderabad (1 Case) • 1997 - Mathews MS et at Vellore (1 Case) • 2003 - Chakrabarti et al Chandigarh (8 Cases) • 2006 - Rao SS, Chandigarh (5 Cases) • 2006 - Jain D, Chandigarh (5 cases) • 2008 - Amit Goyal Lucknow (1 Case) • 2008 - Devi SC Pudducherry (1 Case) • 2008 - Thomas AJ, Vellore (1 Case) • 2008 - Reddy IS Hyderabad© by (1 Case) author • 2010 - Chakrabarti, Chandigarh (16 Cases) • 2010 - Mohapatra, Delhi (1 soil) • ESCMID2010 - Chander, ChandigarhOnline (4 cases)Lecture Library • 2011 - Chander, Chandigarh (1 case) • 2013 - Parsi K, Hyderabad (1 Case) • 2014 – Kulkarni, Mysore (1 case) © by author ESCMID Online Lecture Library Apophysomyces elegans Complex

• The mucoralean fungus Apophysomyces elegans is a thermotolerant species causing severe infections in humans, being the most common site of disease manifestation the cutaneous and subcutaneous tissues, with local invasion into muscle and fat tissue resulting in necrotizing fasciitis • In contrast to the other fungi causing zygomycoses, which have a world-wide distribution and occur only rarely in immunocompetent hosts, A. elegans has been mainly reported in warm climate areas as©an byemerging authorpathogen causing mostly cutaneous infections after injury to the cutaneous barrier • This fungus was discovered in 1979 by Dr. P C Misra and colleaguesESCMIDfrom Indian Onlinesoils and Lectureuntil recently Libraryit was considered the only species of the genus. Now, it is being proposed the genus to have four species i.e. A .elegans, A. trapeziformis, A. ossiformis and A. variabilis Cutaneous Mucormycosis  Cutaneous mucormycosis • This is the third most common form of mucormycosis among humans • It is characterized by necrotizing fasciitis, a soft tissue infection, requiring surgical debridement of necrosed tissue along with antifungals • Disease is found in both immunocompetent and immunocompromised persons, which may prove to be fatal if there is no timely intervention

 Apophysomyces variabilis • Since its discovery, now there are now 4 species of Apophysomyces, namely, A.elegens, A.ossifomis, A.trpaziformis and A.variabilis depending upon the morphology of spores© byand molecularauthorsequence • Out of 4 species of Apophysomyces, A.variabilis is an emerging pathogenic Mucormycete, mainly found prevalent in India • Cause cutaneous, subcutaneous and soft tissue infections following trauma, ESCMIDburns or invasive procedures Onlinein apparently Lecturehealthy hosts Library • A.trapiziformis has recently been reported from soft tissue infections following tornedo in the USA among 13 patients in July 2011 Saksenaea spp.

© by author ESCMID Online Lecture Library Saksenaea vasiformis • First time by Dr. S. B. Saksena in 1953 from India • An infrequent cause of zygomycosis, most often seen in setting of traumatic implantation of fungus • Colonies mature rapidly and are white, cottony and have a white reverse • Sporulation does not occur in routine culture but requires a modified water culture • Sporangiophores are© distinctive,by authorflask -shaped and 24- 64 μm long • RhizoidsESCMIDoften formOnlinenear Lecturebase of sporangiophore, Library are dichotomous and darken as the culture ages Table 2. Clinical profile of patients infected by Saksenaea spp. presented at GMCH, Chandigarh

No. Dates Name / Age / Sex / Anatomical sites Clinical presentations Underlying Risk factors Isolates Outcome Residence involved illnesses

1. 23.11.04 Bachiter Kaur, 65 F, Right gluteal Necrotic patch in the right gluteal None I/M injection on left gluteal S. vasiformis Expired after 2 Ambala (Haryana) region region with surrounding erythema and region 7 days earlier months. induration extending to the anterior abdominal wall

2. 11.11.13 Anita Devi, 31 F, Left gluteal region Large ulcerative lesion (15x10 mm) on None I/M injection on left gluteal S. erythrospora Discharged after 1 Balrampur, (Uttar left gluteal region region 10 days earlier month of Pradesh) admission.

3. 05.04.14 Shabir Ahmed, 37 M, Left shoulder and Necrotizing area on left shoulder None Application of medicated S. erythrospora Discharged after Karnal (Haryana) axilla extending to involve left axillary bandage on the pustule present one and a half region. on the posterior aspect of left month. shoulder.

4. 26.05.14 Anju, 40 F, Karnal Right gluteal Large necrotic area involving whole of None I/M injection over the right S. erythrospora Discharged after (Haryana) region the right© gluteal region.by authorgluteal region 7 days prior one month.

5. 12.06.14 Prem Pal, 60 M, Right gluteal Large necrotic area involving whole of None I/M injection over right gluteal Saksenaea sp. Expired after 2 Karnal (Haryana) region, left gluteal the right gluteal region extending to the region 15 days prior days. ESCMIDregion Onlineleft gluteal region. Lecture Library

6. 23.08.14 Fatima, 55 F, Right gluteal Large ulcerative lesion on right gluteal Diabetes I/M injection on right gluteal Saksenaea sp. Recovering still Samrala (Punjab) region region mellitus region 7 days earlier admitted in the hospital. X-rays Chest : Disseminated Mucormycosis

White mycelial colony with white reverse © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Table 3. Clinical profile of patients infected by Saksenaea spp. published in literature No. Author/year Pt./age/sex Site involved Organism Risk factor Outcome Geog. region 1. Chakrabarti 70 M Anterior S. vasiformis Bull injury Discharged India et al, 1997 abdominal wall 2. Chakrabarti 70 M Prostatectomy S. vasiformis Diabetes Died India et al, 1997 post surgical wound 3. Padmaja et al 35 M Wound in S.vasiformis Postsurgical wound Died India, 2006 right lower infection after Vishakhapatnam quadrant appendectomy 4. Baradkar et 56 F Rhinocerebral S. vasiformis Immunocompetent Died India, Mumbai al, 2008 5. Baradkar et 54 F Left cheek S. vasiformis Immunocompetent Cured India, Mumbai al, 2009 6. Solano et al, 59M Disseminated©S. vasiformisby authorDisseminated in an Died Australia 2000 immunocompetent adult 7. Hospenthal et 26 M Head and S. erythrospora Burns Died Combat al, 2011ESCMIDneck woundsOnline Lecture Library operations in Iraq 8. Lawhon SD, Calf Abomastitis S. erythrospora Premature birth Improved USA et al, 2012 9. Relloso et al, Male - S. erythrospora Sailor accident - Argentine 2014 © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Discussion • Apophysomyces elegans is being reported with increasing frequency as a cause of infection typically in immunocompetent patients following trauma or invasive procedures • Apophysomyces elegans gives an excellent mycelial growth on routine culture media within 24 to 48©hrs bybut authoris notorious for not being able to produce spores ESCMID Online Lecture Library • A simple method of spore induction within 7- 10 days with water and yeast extract (Padhye & Ajello) Discussion • Absidia has certain features similar to Apophysomyces elegans in general morphology with regard to pyriform multispored sporangia arising internodally and not opposite to rhizoids

• The characteristic darkening and thickening of the sporangiophore wall below the apophyses and the characteristic© by funnelauthorshaped apophyses distinctly differentiate fungi of the genus ApophysomycesESCMID Onlineelegans Lecturefrom Libraryother related genera Discussion

• Saksenaea vasiformis resembles Apophysomyces elegans in gross colony morphology and in its inability to sporulate on routine culture media

• The morphology of sporangia on spore induction differentiates© by authorthe two easily ESCMID Online Lecture Library • With the advent of molecular techniques, rapid and accurate identification can easily be done Discussion • Apophysomyces elegans: First isolated by Misra, et al in 1979 from soil samples collected from a mango orchard in northern India. It grows as floccose aerial mycelial in 24 to 48 hours. The apophyses are thick-walled and light brown and have a prominent bell or flask shape or ‘champagne glass’ shaped, feature, differentiating it from Absidia corymbifera, which has less prominent flask-shaped apophyses. • The strains of Apophysomyces have been thought to sporulate with difficulty but in this study all the strains sporulated within 48 hours with their typical appearances without application of special media. • Number of reported cases: Not so many as a total of 74 cases have been reported in the literature. The basic failure lies at the suspicion level, where clinician fails to suspect the fungal etiology of the cutaneous lesions and tries one antibacterial antibiotic after the other. In the meantime lesion becomes so vast that it is unmanageable even if the fungal etiology is established at the terminal stages and patient succumbs to the fungal lesion itself or in combinations with the associated bacterial lesion due to the extensive©skin byinvolvement author. • In the last decade mucormycosis due to A.elegans is being increasingly reported especially from northern parts India from cutaneous as well as invasive lesions • A polyphasic study of clinical and environmental strains of A.elegans, including analysis of severalESCMIDgenes, showed that OnlineA.elegans is a complexLectureof 4 species iLibrary.e. A. elegans, A.ossiformis , A.trapeziformis and A.variabilis. Most of the isolates from India were found to be of A. variabilis clade Discussion contd….. • Infection of cutaneous and subcutaneous tissues with A.elegans is predominantly as a result of the introduction of spore-containing soil and vegetation into wounds arising from trauma or surgery, burns, injection or even an insect bite • Most of the patients had history of associated risk factors. 15 patients had history of injection which developed into local abscess for which incision and drainage was done which eventually led to the classical presentation of cutaneous mucormycosis. • A recent report of A.trapeziformis, wherein cluster of 13 cases of cutaneous mucormycosis has been reported due to A.trapeziformis associated with a Tornado at Joplin, Missouri©in Mayby2011 author, establishing the proposal given by Alvarez et al in 2010 that different clades of Apophysomyces are prevalent in different parts of the world i.e. Indian strains are A.variabilis and American onesESCMIDare A.trapeziformis Online Lecture Library • Aggressive intervention in the form of both surgical debridement and antifungal therapy with liposomal amphotericin B is required to save lives of the patients. Conclusions • The concept of only Apophysomyces elegans and Saksenaea vasiformis has recently changed and now it is a complex of four and three different species, respectively. • Cutaneous mucormycosis due to both is an emerging fungal entity in northern parts of India. • All the strains of Apophysomyces sporulate within 48 hours without using special media either for cultivation or induction of spores and thereby these are identified very easily on morphological grounds. • Other species of Apophysomyces (A.trapeziformis) are also emerging in other parts of world, particularly USA • The main predisposing factors are traumatic injury, iatrogenic (intramuscular injections, incision and drainage without taking aseptic precautions) i.e. those factors which are associated with contact with soil. In Indian patients injection abscess is the highest risk factor leading to cutaneous mucormycosis • It is also involving the immunocompetent© byindividuals, authorwhere there is no obvious underlying factor is elucidated • Most important point to note: A great degree of clinical suspicion and co-ordination of clinician and microbiologist is must for an early diagnosis and proper management of the patients. After wastageESCMIDof substantial time Onlinein not suspecting theLecturefungal etiology, chances Libraryof survival of patient become very bleak • Liposomal amphotericin B, in the form of topical gel as well as intravenous preparations, remains the drug of choice for the ideal outcome of these patients References • Meis JFGM, Kullberg BJ, Pruszczynski M, and R Veth RPH. Severe osteomyelitis due to the zygomycete Apophysomyces elegans. J Clin Microbiol 1994; 32: 3078–81. • Chakrabarti A, Ghosh A, Prasad GS, David JK, Gupta S, Das A, Sakhuja V, Panda NK, Singh SK, Das S, Chakrabarti T. Apophysomyces elegans: An emerging Zygomycete in India. J Clin Microbiol 2003; 41:783-8. • Chander J, Kaur J, Attri A, Mohan H. Primary cutaneous zygomycosis from a tertiary care centre in north-west India. Indian J Med Res. 2010; 131: 765-70. • Chakrabarti A. Cutaneous zygomycosis: major concerns. Indian J Med Res. 2010; 131: 739-41. • Alvarez E, Stchigel AM, Cano J, Sutton DA, Fothergill AW, Chander J, Salas V, Rinaldi MG, Guarro J. Molecular phylogenetic diversity of the emerging mucoralean fungus Apophysomyces: proposal of three new species. Rev Iberoam Micol. 2010; 27: 80-9. • Chakrabarti A, Shivaprakash MR, Curfs-Breuker I, Baghela A, Klaassen CH, Meis JF. Apophysomyces elegans: epidemiology,© by authoramplified fragment length polymorphism typing, and in vitro antifungal susceptibility pattern. J Clin Microbiol. 2010; 48: 4580-5. • Guarro J, Chander J, Alvarez E, Stchigel AM, Kaushik R, Dalal U, Rani H, Punia RS, Cano JF. Apophysomyces variabilis Infections in Humans. Emerg Infect Dis. 2011; 17: 134ESCMID-5. Online Lecture Library • CDC. Notes from the Field: Fatal Fungal Soft-Tissue Infections after a Tornado- Joplin, Missouri. MMWR 2011; 60: 992. Thanks