mycoses
Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Mini-review
Diagnosis of gastrointestinal basidiobolomycosis: a mini-review Mortada H. El-Shabrawi,1 Naglaa Mohamed Kamal,2 Kerstin Kaerger3 and Kerstin Voigt4,5 1 Faculty of Medicine, Cairo University, Cairo, Egypt, 2Faculty of Medicine, Cairo University, Cairo, Egypt, 3Faculty of Biology and Pharmacy, Institute of Microbiology, University of Jena, Jena, Germany, 4Leibniz Institute for Natural Product Research & Infection Biology, Jena, Germany and 5Faculty of Biology and Pharmacy, Institute of Microbiology, University of Jena, Jena, Germany
Summary
Basidiobolus ranarum (Entomophthoromycotina) very rarely affects the gastrointestinal (GI) tract. To date, reported paediatric GI basidiobolomycosis cases are 27 worldwide; 19 from Saudi Arabia and 8 from other parts of the world. Often these cases present a diagnostic dilemma, are prone to misdiagnosis and lack of disease confirmation by proper molecular methodologies. The fungal mass removed by surgery is usually sent for conciliar histopathology, isolation by fungal cultures and final molecular testing for basidiobolomycosis. The incidence of basidiobolomycoses, their predisposing factors and the molecular diagnosis of the fungus causing the disease in combination with a phylogenetic framework are reviewed.
Key words: 18S rRNA, gastrointestinal basidiobolomycosis, identification, molecular typing, Splendore–Hoeppli
phenomenon.
Basidiobolomycosis: main characteristics of a rare chronic subcutaneous disease Basidiobolomycosis is an unusual, rare fungal skin infection causing chronic subcutaneous zygomycosis.1,2 It is caused by Basidiobolus ranarum (Entomophthoromycotina)3,4 with human disease concentrated in tropical and subtropical regions. Extracutaneous involvement is extremely rare5 with gastrointestinal (GI) involvement being exceedingly rare6–10; with only 66 adult and 27 paediatric cases reported worldwide. Most adult cases, 19 patients, were from the United States of whom 17 [89%] were from Arizona11; whereas 14 patients were from Iran,11 12 patients from Iraq,12 11 from the Kingdom of Saudi Arabia (KSA)11 and 4 from Brazil.11 The remaining six patients were one from each of Nigeria, India,
Correspondence: Naglaa Mohamed Kamal, MD, Associate Professor of Pediatrics and Pediatric Hepatology, Faculty of Medicine, Cairo University, Cairo, Egypt. Tel.: +00201141991114. Fax: +20237619012. E-mail: [email protected] or [email protected] Submitted for publication 21 December 2013 Revised 18 June 2014 Accepted for publication 18 June 2014
doi:10.1111/myc.12231
Bangladesh, Italy, Netherlands and one with unreported country of origin.11 The 27 reported paediatric patients are summarised in Table 1,12–24 where 19 patients are from KSA, 3 from Iran, 2 from Iraq, 2 from Brazil and 1 from Nigeria. All reported paediatric gastrointestinal basidiobolomycosis (GIB) cases were males with no significant medical history or apparent predisposing factor(s), age ranged between 1.5–13 years, and presented with fever and abdominal pain as their main symptoms. Leucocytosis with marked eosinophilia, high erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were found in all cases.10,25 Abdominal examination revealed intra-abdominal masses in all cases and were confirmed by abdominal ultrasonography and computed tomography. Almost all cases were misdiagnosed as other chronic granulomatous diseases or malignancies.25 Some examples are: (i) AlJarie series,16 where two patients were misdiagnosed as appendicitis with appendicular mass, two as abdominal tuberculosis and two as lymphomas, (ii) Khan and his colleagues’ patient was also misdiagnosed as intestinal tuberculosis,9 (iii) Fahimzad and his colleagues,17 initially didn’t achieve diagnosis and titled their patient as inflammatory granuloma with undetermined aetiology, (iv) Nguyen’s patient was misdiagnosed as Crohn’s disease,2 etc.
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 138–143
Gastrointestinal basidiobolomycosis
Table 1 Reported paediatric patients of GI basidiobolomycosis.12–24
Reference (year)
Number of reported patients
Edington [13] Bittencourt et al. [14] de Aguiar et al. [15] Al Jarie et al. [16]
1 1 1 6
Male Male Male All males
Fahimzad et al. [17] Hussein et al. [18] El-Shabrawi et al. [19] Arjmand et al. [20] Al-Shanafey et al. [21]
1 1 1 1 9
Male Male Male Male All males
Saadah et al. [22] Alsaleem et al. [23] Zahir et al. [24] Hassan et al. [12]
1 1 1 2
Sex
Male Male Male Both Males
Age (years)
Country of origin
6 13 4 1st: 12 2nd: 12 3rd: 12 4th: 4 5th: 3 6th: 7 1.5 13 10 12 1st: 2 2nd: 12 3rd: 12 4th: 9 5th: 4 6th: 8 7th: 4 8th: 7 9th: 3 2 5 12 1st: 1.5 2nd: 1.5
Nigeria Brazil Brazil KSA
Iran KSA KSA Iran KSA
Molecular identification of basidiobolomycoses
KSA KSA Iran Iraq
In all reported cases, chronic granulomas rich in eosinophils and the Splendore–Hoeppli phenomenon were the usual diagnostic histological criteria.26 Surgical resection and long-term antifungal like amphotericin B were the gold standard treatment in almost all patients.25 A few patients received only medical treatment.16 The outcome was excellent in most cases. Some patients who died were very young and had delayed diagnosis.13,15,16 All the patients who did not receive treatment died.14 First steps of treatment after hospitalisation
Laboratory investigations with close observation are usually requested: complete blood picture with differential counts, CRP, ESR, urinalysis, stool analysis, serum electrolytes, total proteins and albumin, biochemical liver function tests, blood urea nitrogen, serum creatinine and immunological profiles, as well as cultures from blood, urine and stool for bacteria and fungi. Imaging studies, mainly abdominal ultrasonography and computed tomography (CT), are performed.
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 138–143
We had reported one case of GIB from KSA in a 10year-old male child who presented with a tender firm mass in the right iliac fossa, fixed to deep structures confirmed by abdominal imaging involving the caecum with associated marked eosinophilia (17%), thrombocytosis (628 000 mm3), high ESR (39 at 1 h) and high CRP (120 mg/dl).The patient condition rapidly deteriorated with caecal perforation, and right hemicolectomy. Histopathology misdiagnosed it as bilharzial granuloma followed by huge recurrence of the mass, revised histopathology diagnosed basidiobolomycosis with the characteristic Splendore–Hoeppli phenomenon. Long-term antifungal treatment using itraconazole for 1 year was followed by dramatic clinical improvement and regression of the mass with normal follow-up for 3 years.19
Molecular detection of human fungal pathogens, can settle disputed diagnosis in the present era of molecular diagnosis,27 which confirmed the diagnosis in our patient.19 B. ranarum was unequivocally identified using a combination of morphological, physiological and molecular techniques to confirm the infection in the archival formalin-fixed, paraffin-embedded (FFPE) tissue after 6 months of the operation with a protocol which allowed reliable purification of fungal DNA from archival (older than 6 months) FFPE tissue blocks.19 The molecular identification was based on the application of species-specific oligonucleotide primers, Ba1/ Ba2 and Bs1/Bs2, in PCR assays. The extraction protocol omits xylene and uses Roti-Histol as alternative and non-carcinogenic rehydration solvent of the FFPE tissue blocks instead. Extraction of the genomic DNA of the fungal contaminant was performed by the cosurfactant cetyl trimethyl ammonium bromide (CTAB) method adapted for genomic DNA purification from fresh plant tissue. DNA sequencing was performed using the DNA fragments as templates, which were amplified with the taxon-specific primer pairs Ba1/Ba2 (Ba1: 50 -AAAA TCTGTAAGGTTCAACCTTG-30 and Ba2: 50 - TGCAGGA GAAGTACATCCGC- 30 )28 and Bs1/Bs2 (Bs1: 50 -ACT GTTRAMGTATGCTTTGGTAG-30 and Bs2: 50 -CTTGCGA CGCCTCCAACTAG-30 ).27 The primers pair Ba1/Ba2 targets the D1/D2 domain of the nuclear large subunit (28S) ribosomal DNA, whereas the primer pair Bs1/ Bs2 hybridises to the internal transcribed spacer spanning the ITS1-5.8S-ITS2 region of the nuclear ribosomal DNA cluster as reviewed in Rothhardt et al. [27].
139
M. H. El-Shabrawi et al.
The sequences of amplicons are deposited in GenBank under the accession numbers JN201892 and JN201893 for the Bs1/Bs2 and the Ba1/Ba2 PCR fragments for ITS1-5.8S-ITS2 and the D1/D2 domain of 28S rDNA, respectively. The nucleic acid sequences of the 28S and ITS15.8S-ITS1 ribosomal DNA regions were aligned.
equally treated for B. ranarum as they are: Basidiobolus haptosporus, B. heterosporus and B. meristosporus (www.speciesfungorum.org, accessed on 19 Dec 2013).4 (a)
For the 28S rDNA alignment the following sequences were obtained from GenBank and used as reference sequences: JN201893, AB363771, AF113451, AF113452, AF113455, AF113457, AF113458, AJ876792, AY235033, AY546691, DQ273772, DQ273807, DQ364198-207, DQ481224-230, EF392369-429, FJ545245, FN421423, GQ285873-883, HM593512, HM849716, HM849717, JF816213-225, JN131537-542, JN201893, JN939182, JN939188-190, JQ004791-794, JX242591-605, KC146376, NG_027562, NG_027617, NG_027647. For the ITS1-5.8S-ITS2 alignment the following sequences were obtained from GenBank and used as reference sequences: JN943057, EF392524, EF392532, NR_077175, EF392530, EF392519, AY997030, JN201892, EF392540, EF392539, EF392538.
Unweighted distance analyses were carried out on a total of 164 nucleic acid sequences comprising two data sets of 153 28S sequences and 11 ITS sequences. Both data sets were subjected to distance reconstructions using neighbour-Joining (NJ) of Jukes-Cantor distances as implemented in PAUP 4.0b10.29 Bootstrap (BS) proportions30 (50% majority rule) were obtained by 1000 replicates of NJ trees using Jukes-Cantor distances as shown in Fig. 1 and Fig. 2. Basidiobolomycosis was confirmed by molecular and phylogenic analysis.13 Blast searches31 based on the nucleotide sequences revealed 99–100% sequence identity for the Bs1/Bs2 amplicon (JN201892) and 99% sequence identity for the Ba1/Ba2 PCR fragment (JN201893) to B. ranarum confirming the results of the species-specific PCR. From a nomenclatural point of view, there are different synonyms which were
(b)
Figure 1 Phylogenetic relationships within the Entomophtho-
rales based on the neighbour-joining of 1060 aligned nucleotide characters of the nuclear large subunit (28S) ribosomal DNA from a total of 153 taxa. Species of the mucoralean genus Rhizomucor were used as outgroup: (a) higher taxon-rank phylogeny. The Basidiobolus – Schizangiella clade is presented in more detail (b). Basidiobolus ranarum which was deduced from the Ba1/Ba2 LSU fragment (accession number JN201893) is highlighted in bold. Numbers above branches represent bootstrap proportions to survey clade stability support.
140
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 138–143
Gastrointestinal basidiobolomycosis
Figure 2 Phylogenetic relationships within the Basidiobolus – Schizangiella clade based on the 1114 aligned nucleotide characters of the internal transcribed spacer region 1 and 2 disrupted by 5.8S ribosomal DNA (ITS1-5.8S-ITS2) from a total of 11 taxa, 8 ingroup and 3 outgroup taxa from Basidiobolus and Schizangiella, respectively. Numbers above branches represent bootstrap proportions to survey clade stability support.
Therefore, a few Blast hits could be ascribed to these synonymous species designations. The nucleotide sequences from the Ba1/Ba2 (JN201893) and Bs1/Bs2 (JN201892) fragments were embedded in single locus sets of reference sequences for 28S and ITS1-5.8S-ITS2 loci obtained from GenBank (http://www.ncbi.nlm.nih. gov/ accessed on 19 Dec 2013) aligned and subjected to phylogenetic analyses, which are shown in Fig. 1 and Fig. 2, respectively for each data set. The nucleotide sequence of Ba1/Ba2 (JN201893) revealed unequivocal classification of the causative agent of the GIB within the Basidiobolus clade to B. ranarum (Fig. 1). The genus Schizangiella appeared as the closest related genus to Basidiobolus (Fig. 1). Closest relative of the causative agent of GIB was B. ranarum NRRL20525 (Fig. 1b). At the ITS1-5.8S-ITS2 level the causative agent of GI basidiobolomycosis grouped basal to the B. ranarum core group (Fig. 2). By this way diagnosis of B. ranarum was confirmed by molecular and phylogenetic analyses.
Discussion Basidiobolus ranarum is a known cause of chronic subcutaneous zygomycosis. During the past decade, many cases have been reported with extracutaneous basidiobolomycosis. GI basidiobolomycosis is rare but
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 138–143
emerging fungal infection causing serious, and occasionally fatal, paediatric disease.25 Surveying the worldwide cases of basidiobolomycoses male children seem to be more frequently afflicted, a hypothesis which is in agreement with the findings by Pfaller and Diekema [32] and Ribes et al [26]. The main differential diagnosis of GIB with granuloma includes inflammatory bowel disease, intestinal tuberculosis, sarcoidosis, amebiasis and malignancy.19 The diagnosis of GIB is always confusing and requires a high index of suspicion.15 So far, there is no wellidentified risk factor. However, the diagnosis might be suspected in the previously healthy children, especially those living in, or near, tropical areas who develop symptoms that may suggest the diagnosis.23 To our knowledge all the reported cases were diagnosed based on the histologic findings of the resected masses and we were the first group who reported confirming the diagnosis by molecular testing for basidiobolomycosis in the FFPE intestinal tissue by ribosomal DNA sequencing.19 The molecular identification of invasive human pathogenic fungi is a tedious task. There have been cases with discrepant histologic, culture and molecular taxonomic results at final diagnosis resulting from the decreasing quality of archival FFPE tissues. Such discrepancies could lead to unnecessary pharmaceutical exposure and/or inappropriate treatment.33–34 Therefore, our efforts to improve the sensitivity and specificity of diagnostic tests need to be increased in order aim a straight forward and unequivocal polyphasic diagnosis which involves histologic and culture-dependent methods confirmed by cultivationindependent molecular identification. Reviewing literature since the publication of our report up till present time revealed that no other authors have used molecular identification in GIB identification, that urged us to present the molecular technique in details aiming to encourage other researchers to use the presented protocol which allows reliable purification of fungal DNA from archival FFPE tissue blocks. A reliable procedure like this may open the door for researchers who feel they had at a time a case suspected of these neglected fungal infections, to use the described technique to retrospectively work the FFPE tissues of their patients. The aim is to uncover the actual magnitude of neglected basidiobolomycotic fungal infection, which although is endemic in certain tropical areas like Uganda, certain areas of Africa, India and other parts of Asia,1 but is found worldwide, even in areas where the disease has not been yet reported.
141
M. H. El-Shabrawi et al.
Conclusion Molecular testing of basidiobolomycosis might prove to be the most accurate method to prove diagnosis. Elucidation of infection in FFPE intestinal tissue by ribosomal DNA sequencing can precisely confirm the diagnosis in archived specimens. In the present era of molecular diagnosis, further researches concerning molecular detection of human fungal pathogens are urged as they can definitely settle disputed diagnosis.
Acknowledgments
9
10
11
12
13
The authors thank Domenica Schnabelrauch (MPI Chemical Ecology Jena, Germany) for technical assistance in DNA sequencing. KV wishes to thank Prof. Rolf Beutel and Lars M€ ockel (Institute of Systematic Zoology and Evolutionary Biology, University of Jena, Germany) for many inspiring discussions on the evolution of Entomophthorales leading to the establishment of the set of reference sequences. This work was financially supported by the Deutsche Forschungsgemeinschaft (DFG) within CRC/TR 124 FungiNet: project Z1 to KV. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
14
Conflicts of interest
20
Authors declare no conflicts of interest.
21
References
22
1
2 3 4
5 6
7
8
142
Sugar AM. Agents of mucormycosis and related species. In: Mandell GL, Bennett JE, Dolin R (eds), Mandell, Douglas and Bennett’s Principles and Practice of Infectious Diseases. New York: Churchill Livingstone, 1995: 4th edn, vol. 2, 2311–21. Nguyen BD. CT features of basidiobolomycosis with gastrointestinal and urinary involvement. AJR Am J Roentgenol 2000; 174: 878–9. Hibbett DS, Binder M, Bischoff JF et al. A higher-level phylogenetic classification of the Fungi. Mycol Res 2007; 111: 509–47. Kirk PM, Cannon PF, Minter DW, Stalpers JA. Ainsworth & Bisby’s Dictionary of the Fungi. 10th ed. Wallingford, UK: CAB International, CSIRO Publishing, 2008. Kwon-Chung KJ, Bennett JE. Medical Mycology. Philadelphia: Lea & Febiger, 1992: 449–63. Centers for Disease Control and Prevention. Gastrointestinal basidiobolomycosis-Arizona, 1994–1999. MMWR Morb Mortal Wkly Rep 1999; 48: 710–3. Yousef OM, Smilack JD, Kerr DM, Ramsey R, Rosati L, Colby TV. Gastrointestinal basidiobolomycosis. Morphologic findings in a cluster of six cases. Am J Clin Pathol 1999; 112: 610–6. Zavasky DM, Samowitz W, Loftus T, Segal H, Carroll K. Gastrointestinal zygomycotic infection caused by Basidiobolus ranarum: case report and review. Clin Infect Dis 1999; 28: 1244–8.
15
16 17
18
19
23
24
25
26 27
28
Khan ZU, Khoursheed M, Makar R et al. Basidiobolus ranarum as an etiologic agent of gastrointestinal zygomycosis. J Clin Microbiol 2001; 39: 2360–3. Lyon GM, Smilack JD, Komatsu KK et al. Gastrointestinal basidiobolomycosis in Arizona: clinical and epidemiological characteristics and review of the literature. Clin Infect Dis 2001; 32: 1448–55. Geramizadeh B, Foroughi R, Keshtkar-Jahromi M, Malek-Hosseini SA, Alborzi A. Gastrointestinal basidiobolomycosis, an emerging infection in the immunocompetent host: a report of 14 patients. J Med Microbiol 2012; 61(Pt 12): 1770–4. doi:10.1099/jmm.0. 046839-0. Hassan HA, Majid RA, Rashid NG et al. Eosinophilic granulomatous gastrointestinal and hepatic abscesses attributable tobasidiobolomycosis and fasciolias: a simultaneous emergence in Iraqi Kurdistan. BMC Infect Dis 2013; 13: 91. doi:10.1186/ 1471-2334-13-91. Edington GM. Phycomycosis in Ibadan, Western Nigeria: two post-mortem reports. Trans R Soc Trop Med Hyg 1964; 58: 242–5. Bittencourt AL, Londero AT, Araujo MS, Mendonca N, Bastos JL. Occurrence of subcutaneous zygomycosis caused by Basidiobolus haptosporus in Brazil. Mycopathologia 1979; 68: 101–4. de Aguiar E, Moraes WC, Londero AT. Gastrointestinal entomophthoramycosis caused by Basidiobolus haptosporus. Mycopathologia 1980; 72: 101–5. Al Jarie A, Al-Mohsen I, Al Jumaah S et al. Pediatric gastrointestinal basidiobolomycosis. Pediatr Infect Dis J 2003; 22: 1007–14. Fahimzad A, Karimi A, Tabatabaei SR, Zadeh MG. Gastrointestinal basidiobolomycosis as a rare etiology of bowel obstruction. Turk J Med Sci 2006; 36: 239–41. Hussein MR, Musalam AO, Assiry MH, Eid RA, El Motawa AM, Gamel AM. Histological and ultrastructural features of gastrointestinal basidiobolomycosis. Mycol Res 2007; 111: 926–30. El-Shabrawi MH, Kamal NM, Jouini R, Al-Harbi A, Voigt K, Al-Malki T. Gastrointestinal basidiobolomycosis: an emerging fungal infection causing bowel perforation in a child. J Med Microbiol 2011; 60(Pt 9): 1395–402. doi: 10.1099/jmm.0.028613-0. Epub 2011 May 12. PMID: 21566088 Arjmand R, Karimi A, Sanaei Dashti A, Kadivar M. A child with intestinal basidiobolomycosis. Iran J Med Sci 2012; 37: 134–6. Al-Shanafey S, AlRobean F, Bin Hussain I. Surgical management of gastrointestinal basidiobolomycosis in pediatric patients. J Pediatr Surg 2012; 47: 949–51. doi:10.1016/j.jpedsurg.2012.01.053. Saadah OI, Farouq MF, Daajani NA, Kamal JS, Ghanem AT. Gastrointestinal basidiobolomycosis in a child; an unusual fungal infection mimicking fistulising Crohn’s disease. J Crohns Colitis 2012; 6: 368– 72. doi:10.1016/j.crohns.10.008. Alsaleem K, Al-Mehaidib A, Banemai M, Bin-Hussain I, Faqih M, Mehmadi AA. Gastrointestinal basidiobolomycosis: mimicking Crohns disease case report and review of the literature. Ann Saudi Med 2013; 33: 500–4. doi:10.5144/0256-4947.2013.500. Zahir ST, Sharahjin NS, Kargar S. Basidiobolomycosis a mysterious fungal infection mimic small intestinal and colonic tumour with renal insufficiency and ominous outcome. BMJ Case Rep 2013; 26. pii: bcr2013200244. doi: 10.1136/bcr-2013-200244. El-Shabrawi MH, Kamal NM. Gastrointestinal basidiobolomycosis in children: an overlooked emerging infection? J Med Microbiol 2011; 60(Pt 7): 871–80. Ribes JA, Vanover-Sams CL, Baker DJ. Zygomycetes in human disease. Clin Microbiol Rev 2000; 13: 236–301. Rothhardt JE, Schwartze VU, Voigt K. Entomophthorales. In: Dongyou L. (ed.) Molecular Detection of Human Fungal Pathogens. New York: Taylor and Francis CRC Press, 2011: Chapter 83, 709–20. Voigt K, Cigelnik E, O0 Donnell K. Phylogeny and PCR identification of clinically important zygomycetes based on nuclear ribosomal-DNA sequence data. J Clin Microbiol 1999; 37: 3957–64.
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 138–143
Gastrointestinal basidiobolomycosis
29
30 31 32
Swofford DL. PAUP*: Phylogenetic Analysis Using Parsimony (*and other methods). Version 4. Sunderland, MA: Sinauer Associates, 1998. Felsenstein J. Confidence limits of phylogenies: an approach using the bootstrap. Evolution 1985; 39: 783–91. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215: 403–10. Pfaller MA, Diekema DJ. Unusual fungal and pseudofungal infections of humans. J Clin Microbiol 2005; 43: 1495–504.
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 138–143
33
34
Lopez FA, Crowley RS, Wastila L, Valantine HA, Remington JS. Scedosporium apiospermum (Pseudallescheria boydii) infection in a heart transplant recipient: a case of mistaken identity. J Heart Lung Transplant 1998; 17: 321–4. Sangoi AR, Rogers WM, Longacre TA, Montoya JG, Baron EJ, Banaei N. Challenges and pitfalls of morphologic identification of fungal infections in histologic and cytologic specimens: a ten-year retrospective review at a single institution. Am J Clin Pathol 2009; 131: 364– 75.
143
Copyright of Mycoses is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Mini-review
Diagnosis of gastrointestinal basidiobolomycosis: a mini-review Mortada H. El-Shabrawi,1 Naglaa Mohamed Kamal,2 Kerstin Kaerger3 and Kerstin Voigt4,5 1 Faculty of Medicine, Cairo University, Cairo, Egypt, 2Faculty of Medicine, Cairo University, Cairo, Egypt, 3Faculty of Biology and Pharmacy, Institute of Microbiology, University of Jena, Jena, Germany, 4Leibniz Institute for Natural Product Research & Infection Biology, Jena, Germany and 5Faculty of Biology and Pharmacy, Institute of Microbiology, University of Jena, Jena, Germany
Summary
Basidiobolus ranarum (Entomophthoromycotina) very rarely affects the gastrointestinal (GI) tract. To date, reported paediatric GI basidiobolomycosis cases are 27 worldwide; 19 from Saudi Arabia and 8 from other parts of the world. Often these cases present a diagnostic dilemma, are prone to misdiagnosis and lack of disease confirmation by proper molecular methodologies. The fungal mass removed by surgery is usually sent for conciliar histopathology, isolation by fungal cultures and final molecular testing for basidiobolomycosis. The incidence of basidiobolomycoses, their predisposing factors and the molecular diagnosis of the fungus causing the disease in combination with a phylogenetic framework are reviewed.
Key words: 18S rRNA, gastrointestinal basidiobolomycosis, identification, molecular typing, Splendore–Hoeppli
phenomenon.
Basidiobolomycosis: main characteristics of a rare chronic subcutaneous disease Basidiobolomycosis is an unusual, rare fungal skin infection causing chronic subcutaneous zygomycosis.1,2 It is caused by Basidiobolus ranarum (Entomophthoromycotina)3,4 with human disease concentrated in tropical and subtropical regions. Extracutaneous involvement is extremely rare5 with gastrointestinal (GI) involvement being exceedingly rare6–10; with only 66 adult and 27 paediatric cases reported worldwide. Most adult cases, 19 patients, were from the United States of whom 17 [89%] were from Arizona11; whereas 14 patients were from Iran,11 12 patients from Iraq,12 11 from the Kingdom of Saudi Arabia (KSA)11 and 4 from Brazil.11 The remaining six patients were one from each of Nigeria, India,
Correspondence: Naglaa Mohamed Kamal, MD, Associate Professor of Pediatrics and Pediatric Hepatology, Faculty of Medicine, Cairo University, Cairo, Egypt. Tel.: +00201141991114. Fax: +20237619012. E-mail: [email protected] or [email protected] Submitted for publication 21 December 2013 Revised 18 June 2014 Accepted for publication 18 June 2014
doi:10.1111/myc.12231
Bangladesh, Italy, Netherlands and one with unreported country of origin.11 The 27 reported paediatric patients are summarised in Table 1,12–24 where 19 patients are from KSA, 3 from Iran, 2 from Iraq, 2 from Brazil and 1 from Nigeria. All reported paediatric gastrointestinal basidiobolomycosis (GIB) cases were males with no significant medical history or apparent predisposing factor(s), age ranged between 1.5–13 years, and presented with fever and abdominal pain as their main symptoms. Leucocytosis with marked eosinophilia, high erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were found in all cases.10,25 Abdominal examination revealed intra-abdominal masses in all cases and were confirmed by abdominal ultrasonography and computed tomography. Almost all cases were misdiagnosed as other chronic granulomatous diseases or malignancies.25 Some examples are: (i) AlJarie series,16 where two patients were misdiagnosed as appendicitis with appendicular mass, two as abdominal tuberculosis and two as lymphomas, (ii) Khan and his colleagues’ patient was also misdiagnosed as intestinal tuberculosis,9 (iii) Fahimzad and his colleagues,17 initially didn’t achieve diagnosis and titled their patient as inflammatory granuloma with undetermined aetiology, (iv) Nguyen’s patient was misdiagnosed as Crohn’s disease,2 etc.
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 138–143
Gastrointestinal basidiobolomycosis
Table 1 Reported paediatric patients of GI basidiobolomycosis.12–24
Reference (year)
Number of reported patients
Edington [13] Bittencourt et al. [14] de Aguiar et al. [15] Al Jarie et al. [16]
1 1 1 6
Male Male Male All males
Fahimzad et al. [17] Hussein et al. [18] El-Shabrawi et al. [19] Arjmand et al. [20] Al-Shanafey et al. [21]
1 1 1 1 9
Male Male Male Male All males
Saadah et al. [22] Alsaleem et al. [23] Zahir et al. [24] Hassan et al. [12]
1 1 1 2
Sex
Male Male Male Both Males
Age (years)
Country of origin
6 13 4 1st: 12 2nd: 12 3rd: 12 4th: 4 5th: 3 6th: 7 1.5 13 10 12 1st: 2 2nd: 12 3rd: 12 4th: 9 5th: 4 6th: 8 7th: 4 8th: 7 9th: 3 2 5 12 1st: 1.5 2nd: 1.5
Nigeria Brazil Brazil KSA
Iran KSA KSA Iran KSA
Molecular identification of basidiobolomycoses
KSA KSA Iran Iraq
In all reported cases, chronic granulomas rich in eosinophils and the Splendore–Hoeppli phenomenon were the usual diagnostic histological criteria.26 Surgical resection and long-term antifungal like amphotericin B were the gold standard treatment in almost all patients.25 A few patients received only medical treatment.16 The outcome was excellent in most cases. Some patients who died were very young and had delayed diagnosis.13,15,16 All the patients who did not receive treatment died.14 First steps of treatment after hospitalisation
Laboratory investigations with close observation are usually requested: complete blood picture with differential counts, CRP, ESR, urinalysis, stool analysis, serum electrolytes, total proteins and albumin, biochemical liver function tests, blood urea nitrogen, serum creatinine and immunological profiles, as well as cultures from blood, urine and stool for bacteria and fungi. Imaging studies, mainly abdominal ultrasonography and computed tomography (CT), are performed.
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 138–143
We had reported one case of GIB from KSA in a 10year-old male child who presented with a tender firm mass in the right iliac fossa, fixed to deep structures confirmed by abdominal imaging involving the caecum with associated marked eosinophilia (17%), thrombocytosis (628 000 mm3), high ESR (39 at 1 h) and high CRP (120 mg/dl).The patient condition rapidly deteriorated with caecal perforation, and right hemicolectomy. Histopathology misdiagnosed it as bilharzial granuloma followed by huge recurrence of the mass, revised histopathology diagnosed basidiobolomycosis with the characteristic Splendore–Hoeppli phenomenon. Long-term antifungal treatment using itraconazole for 1 year was followed by dramatic clinical improvement and regression of the mass with normal follow-up for 3 years.19
Molecular detection of human fungal pathogens, can settle disputed diagnosis in the present era of molecular diagnosis,27 which confirmed the diagnosis in our patient.19 B. ranarum was unequivocally identified using a combination of morphological, physiological and molecular techniques to confirm the infection in the archival formalin-fixed, paraffin-embedded (FFPE) tissue after 6 months of the operation with a protocol which allowed reliable purification of fungal DNA from archival (older than 6 months) FFPE tissue blocks.19 The molecular identification was based on the application of species-specific oligonucleotide primers, Ba1/ Ba2 and Bs1/Bs2, in PCR assays. The extraction protocol omits xylene and uses Roti-Histol as alternative and non-carcinogenic rehydration solvent of the FFPE tissue blocks instead. Extraction of the genomic DNA of the fungal contaminant was performed by the cosurfactant cetyl trimethyl ammonium bromide (CTAB) method adapted for genomic DNA purification from fresh plant tissue. DNA sequencing was performed using the DNA fragments as templates, which were amplified with the taxon-specific primer pairs Ba1/Ba2 (Ba1: 50 -AAAA TCTGTAAGGTTCAACCTTG-30 and Ba2: 50 - TGCAGGA GAAGTACATCCGC- 30 )28 and Bs1/Bs2 (Bs1: 50 -ACT GTTRAMGTATGCTTTGGTAG-30 and Bs2: 50 -CTTGCGA CGCCTCCAACTAG-30 ).27 The primers pair Ba1/Ba2 targets the D1/D2 domain of the nuclear large subunit (28S) ribosomal DNA, whereas the primer pair Bs1/ Bs2 hybridises to the internal transcribed spacer spanning the ITS1-5.8S-ITS2 region of the nuclear ribosomal DNA cluster as reviewed in Rothhardt et al. [27].
139
M. H. El-Shabrawi et al.
The sequences of amplicons are deposited in GenBank under the accession numbers JN201892 and JN201893 for the Bs1/Bs2 and the Ba1/Ba2 PCR fragments for ITS1-5.8S-ITS2 and the D1/D2 domain of 28S rDNA, respectively. The nucleic acid sequences of the 28S and ITS15.8S-ITS1 ribosomal DNA regions were aligned.
equally treated for B. ranarum as they are: Basidiobolus haptosporus, B. heterosporus and B. meristosporus (www.speciesfungorum.org, accessed on 19 Dec 2013).4 (a)
For the 28S rDNA alignment the following sequences were obtained from GenBank and used as reference sequences: JN201893, AB363771, AF113451, AF113452, AF113455, AF113457, AF113458, AJ876792, AY235033, AY546691, DQ273772, DQ273807, DQ364198-207, DQ481224-230, EF392369-429, FJ545245, FN421423, GQ285873-883, HM593512, HM849716, HM849717, JF816213-225, JN131537-542, JN201893, JN939182, JN939188-190, JQ004791-794, JX242591-605, KC146376, NG_027562, NG_027617, NG_027647. For the ITS1-5.8S-ITS2 alignment the following sequences were obtained from GenBank and used as reference sequences: JN943057, EF392524, EF392532, NR_077175, EF392530, EF392519, AY997030, JN201892, EF392540, EF392539, EF392538.
Unweighted distance analyses were carried out on a total of 164 nucleic acid sequences comprising two data sets of 153 28S sequences and 11 ITS sequences. Both data sets were subjected to distance reconstructions using neighbour-Joining (NJ) of Jukes-Cantor distances as implemented in PAUP 4.0b10.29 Bootstrap (BS) proportions30 (50% majority rule) were obtained by 1000 replicates of NJ trees using Jukes-Cantor distances as shown in Fig. 1 and Fig. 2. Basidiobolomycosis was confirmed by molecular and phylogenic analysis.13 Blast searches31 based on the nucleotide sequences revealed 99–100% sequence identity for the Bs1/Bs2 amplicon (JN201892) and 99% sequence identity for the Ba1/Ba2 PCR fragment (JN201893) to B. ranarum confirming the results of the species-specific PCR. From a nomenclatural point of view, there are different synonyms which were
(b)
Figure 1 Phylogenetic relationships within the Entomophtho-
rales based on the neighbour-joining of 1060 aligned nucleotide characters of the nuclear large subunit (28S) ribosomal DNA from a total of 153 taxa. Species of the mucoralean genus Rhizomucor were used as outgroup: (a) higher taxon-rank phylogeny. The Basidiobolus – Schizangiella clade is presented in more detail (b). Basidiobolus ranarum which was deduced from the Ba1/Ba2 LSU fragment (accession number JN201893) is highlighted in bold. Numbers above branches represent bootstrap proportions to survey clade stability support.
140
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 138–143
Gastrointestinal basidiobolomycosis
Figure 2 Phylogenetic relationships within the Basidiobolus – Schizangiella clade based on the 1114 aligned nucleotide characters of the internal transcribed spacer region 1 and 2 disrupted by 5.8S ribosomal DNA (ITS1-5.8S-ITS2) from a total of 11 taxa, 8 ingroup and 3 outgroup taxa from Basidiobolus and Schizangiella, respectively. Numbers above branches represent bootstrap proportions to survey clade stability support.
Therefore, a few Blast hits could be ascribed to these synonymous species designations. The nucleotide sequences from the Ba1/Ba2 (JN201893) and Bs1/Bs2 (JN201892) fragments were embedded in single locus sets of reference sequences for 28S and ITS1-5.8S-ITS2 loci obtained from GenBank (http://www.ncbi.nlm.nih. gov/ accessed on 19 Dec 2013) aligned and subjected to phylogenetic analyses, which are shown in Fig. 1 and Fig. 2, respectively for each data set. The nucleotide sequence of Ba1/Ba2 (JN201893) revealed unequivocal classification of the causative agent of the GIB within the Basidiobolus clade to B. ranarum (Fig. 1). The genus Schizangiella appeared as the closest related genus to Basidiobolus (Fig. 1). Closest relative of the causative agent of GIB was B. ranarum NRRL20525 (Fig. 1b). At the ITS1-5.8S-ITS2 level the causative agent of GI basidiobolomycosis grouped basal to the B. ranarum core group (Fig. 2). By this way diagnosis of B. ranarum was confirmed by molecular and phylogenetic analyses.
Discussion Basidiobolus ranarum is a known cause of chronic subcutaneous zygomycosis. During the past decade, many cases have been reported with extracutaneous basidiobolomycosis. GI basidiobolomycosis is rare but
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 138–143
emerging fungal infection causing serious, and occasionally fatal, paediatric disease.25 Surveying the worldwide cases of basidiobolomycoses male children seem to be more frequently afflicted, a hypothesis which is in agreement with the findings by Pfaller and Diekema [32] and Ribes et al [26]. The main differential diagnosis of GIB with granuloma includes inflammatory bowel disease, intestinal tuberculosis, sarcoidosis, amebiasis and malignancy.19 The diagnosis of GIB is always confusing and requires a high index of suspicion.15 So far, there is no wellidentified risk factor. However, the diagnosis might be suspected in the previously healthy children, especially those living in, or near, tropical areas who develop symptoms that may suggest the diagnosis.23 To our knowledge all the reported cases were diagnosed based on the histologic findings of the resected masses and we were the first group who reported confirming the diagnosis by molecular testing for basidiobolomycosis in the FFPE intestinal tissue by ribosomal DNA sequencing.19 The molecular identification of invasive human pathogenic fungi is a tedious task. There have been cases with discrepant histologic, culture and molecular taxonomic results at final diagnosis resulting from the decreasing quality of archival FFPE tissues. Such discrepancies could lead to unnecessary pharmaceutical exposure and/or inappropriate treatment.33–34 Therefore, our efforts to improve the sensitivity and specificity of diagnostic tests need to be increased in order aim a straight forward and unequivocal polyphasic diagnosis which involves histologic and culture-dependent methods confirmed by cultivationindependent molecular identification. Reviewing literature since the publication of our report up till present time revealed that no other authors have used molecular identification in GIB identification, that urged us to present the molecular technique in details aiming to encourage other researchers to use the presented protocol which allows reliable purification of fungal DNA from archival FFPE tissue blocks. A reliable procedure like this may open the door for researchers who feel they had at a time a case suspected of these neglected fungal infections, to use the described technique to retrospectively work the FFPE tissues of their patients. The aim is to uncover the actual magnitude of neglected basidiobolomycotic fungal infection, which although is endemic in certain tropical areas like Uganda, certain areas of Africa, India and other parts of Asia,1 but is found worldwide, even in areas where the disease has not been yet reported.
141
M. H. El-Shabrawi et al.
Conclusion Molecular testing of basidiobolomycosis might prove to be the most accurate method to prove diagnosis. Elucidation of infection in FFPE intestinal tissue by ribosomal DNA sequencing can precisely confirm the diagnosis in archived specimens. In the present era of molecular diagnosis, further researches concerning molecular detection of human fungal pathogens are urged as they can definitely settle disputed diagnosis.
Acknowledgments
9
10
11
12
13
The authors thank Domenica Schnabelrauch (MPI Chemical Ecology Jena, Germany) for technical assistance in DNA sequencing. KV wishes to thank Prof. Rolf Beutel and Lars M€ ockel (Institute of Systematic Zoology and Evolutionary Biology, University of Jena, Germany) for many inspiring discussions on the evolution of Entomophthorales leading to the establishment of the set of reference sequences. This work was financially supported by the Deutsche Forschungsgemeinschaft (DFG) within CRC/TR 124 FungiNet: project Z1 to KV. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
14
Conflicts of interest
20
Authors declare no conflicts of interest.
21
References
22
1
2 3 4
5 6
7
8
142
Sugar AM. Agents of mucormycosis and related species. In: Mandell GL, Bennett JE, Dolin R (eds), Mandell, Douglas and Bennett’s Principles and Practice of Infectious Diseases. New York: Churchill Livingstone, 1995: 4th edn, vol. 2, 2311–21. Nguyen BD. CT features of basidiobolomycosis with gastrointestinal and urinary involvement. AJR Am J Roentgenol 2000; 174: 878–9. Hibbett DS, Binder M, Bischoff JF et al. A higher-level phylogenetic classification of the Fungi. Mycol Res 2007; 111: 509–47. Kirk PM, Cannon PF, Minter DW, Stalpers JA. Ainsworth & Bisby’s Dictionary of the Fungi. 10th ed. Wallingford, UK: CAB International, CSIRO Publishing, 2008. Kwon-Chung KJ, Bennett JE. Medical Mycology. Philadelphia: Lea & Febiger, 1992: 449–63. Centers for Disease Control and Prevention. Gastrointestinal basidiobolomycosis-Arizona, 1994–1999. MMWR Morb Mortal Wkly Rep 1999; 48: 710–3. Yousef OM, Smilack JD, Kerr DM, Ramsey R, Rosati L, Colby TV. Gastrointestinal basidiobolomycosis. Morphologic findings in a cluster of six cases. Am J Clin Pathol 1999; 112: 610–6. Zavasky DM, Samowitz W, Loftus T, Segal H, Carroll K. Gastrointestinal zygomycotic infection caused by Basidiobolus ranarum: case report and review. Clin Infect Dis 1999; 28: 1244–8.
15
16 17
18
19
23
24
25
26 27
28
Khan ZU, Khoursheed M, Makar R et al. Basidiobolus ranarum as an etiologic agent of gastrointestinal zygomycosis. J Clin Microbiol 2001; 39: 2360–3. Lyon GM, Smilack JD, Komatsu KK et al. Gastrointestinal basidiobolomycosis in Arizona: clinical and epidemiological characteristics and review of the literature. Clin Infect Dis 2001; 32: 1448–55. Geramizadeh B, Foroughi R, Keshtkar-Jahromi M, Malek-Hosseini SA, Alborzi A. Gastrointestinal basidiobolomycosis, an emerging infection in the immunocompetent host: a report of 14 patients. J Med Microbiol 2012; 61(Pt 12): 1770–4. doi:10.1099/jmm.0. 046839-0. Hassan HA, Majid RA, Rashid NG et al. Eosinophilic granulomatous gastrointestinal and hepatic abscesses attributable tobasidiobolomycosis and fasciolias: a simultaneous emergence in Iraqi Kurdistan. BMC Infect Dis 2013; 13: 91. doi:10.1186/ 1471-2334-13-91. Edington GM. Phycomycosis in Ibadan, Western Nigeria: two post-mortem reports. Trans R Soc Trop Med Hyg 1964; 58: 242–5. Bittencourt AL, Londero AT, Araujo MS, Mendonca N, Bastos JL. Occurrence of subcutaneous zygomycosis caused by Basidiobolus haptosporus in Brazil. Mycopathologia 1979; 68: 101–4. de Aguiar E, Moraes WC, Londero AT. Gastrointestinal entomophthoramycosis caused by Basidiobolus haptosporus. Mycopathologia 1980; 72: 101–5. Al Jarie A, Al-Mohsen I, Al Jumaah S et al. Pediatric gastrointestinal basidiobolomycosis. Pediatr Infect Dis J 2003; 22: 1007–14. Fahimzad A, Karimi A, Tabatabaei SR, Zadeh MG. Gastrointestinal basidiobolomycosis as a rare etiology of bowel obstruction. Turk J Med Sci 2006; 36: 239–41. Hussein MR, Musalam AO, Assiry MH, Eid RA, El Motawa AM, Gamel AM. Histological and ultrastructural features of gastrointestinal basidiobolomycosis. Mycol Res 2007; 111: 926–30. El-Shabrawi MH, Kamal NM, Jouini R, Al-Harbi A, Voigt K, Al-Malki T. Gastrointestinal basidiobolomycosis: an emerging fungal infection causing bowel perforation in a child. J Med Microbiol 2011; 60(Pt 9): 1395–402. doi: 10.1099/jmm.0.028613-0. Epub 2011 May 12. PMID: 21566088 Arjmand R, Karimi A, Sanaei Dashti A, Kadivar M. A child with intestinal basidiobolomycosis. Iran J Med Sci 2012; 37: 134–6. Al-Shanafey S, AlRobean F, Bin Hussain I. Surgical management of gastrointestinal basidiobolomycosis in pediatric patients. J Pediatr Surg 2012; 47: 949–51. doi:10.1016/j.jpedsurg.2012.01.053. Saadah OI, Farouq MF, Daajani NA, Kamal JS, Ghanem AT. Gastrointestinal basidiobolomycosis in a child; an unusual fungal infection mimicking fistulising Crohn’s disease. J Crohns Colitis 2012; 6: 368– 72. doi:10.1016/j.crohns.10.008. Alsaleem K, Al-Mehaidib A, Banemai M, Bin-Hussain I, Faqih M, Mehmadi AA. Gastrointestinal basidiobolomycosis: mimicking Crohns disease case report and review of the literature. Ann Saudi Med 2013; 33: 500–4. doi:10.5144/0256-4947.2013.500. Zahir ST, Sharahjin NS, Kargar S. Basidiobolomycosis a mysterious fungal infection mimic small intestinal and colonic tumour with renal insufficiency and ominous outcome. BMJ Case Rep 2013; 26. pii: bcr2013200244. doi: 10.1136/bcr-2013-200244. El-Shabrawi MH, Kamal NM. Gastrointestinal basidiobolomycosis in children: an overlooked emerging infection? J Med Microbiol 2011; 60(Pt 7): 871–80. Ribes JA, Vanover-Sams CL, Baker DJ. Zygomycetes in human disease. Clin Microbiol Rev 2000; 13: 236–301. Rothhardt JE, Schwartze VU, Voigt K. Entomophthorales. In: Dongyou L. (ed.) Molecular Detection of Human Fungal Pathogens. New York: Taylor and Francis CRC Press, 2011: Chapter 83, 709–20. Voigt K, Cigelnik E, O0 Donnell K. Phylogeny and PCR identification of clinically important zygomycetes based on nuclear ribosomal-DNA sequence data. J Clin Microbiol 1999; 37: 3957–64.
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 138–143
Gastrointestinal basidiobolomycosis
29
30 31 32
Swofford DL. PAUP*: Phylogenetic Analysis Using Parsimony (*and other methods). Version 4. Sunderland, MA: Sinauer Associates, 1998. Felsenstein J. Confidence limits of phylogenies: an approach using the bootstrap. Evolution 1985; 39: 783–91. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215: 403–10. Pfaller MA, Diekema DJ. Unusual fungal and pseudofungal infections of humans. J Clin Microbiol 2005; 43: 1495–504.
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57 (Suppl. 3), 138–143
33
34
Lopez FA, Crowley RS, Wastila L, Valantine HA, Remington JS. Scedosporium apiospermum (Pseudallescheria boydii) infection in a heart transplant recipient: a case of mistaken identity. J Heart Lung Transplant 1998; 17: 321–4. Sangoi AR, Rogers WM, Longacre TA, Montoya JG, Baron EJ, Banaei N. Challenges and pitfalls of morphologic identification of fungal infections in histologic and cytologic specimens: a ten-year retrospective review at a single institution. Am J Clin Pathol 2009; 131: 364– 75.
143
Copyright of Mycoses is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
