Medical Mycology Case Reports 2 (2013) 44–47
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Medical Mycology Case Reports journal homepage: www.elsevier.com/locate/mmcr
A disseminated infection with the antifungal-multiresistant teleomorphic fungus Neocosmospora vasinfecta in a patient with acute B-lymphoblastic leukemia Fre´de´ric Gabriel a,b,c, Mahussi D’Almeida a,b,c, Olivier Albert a,b,c, Vale´rie Fitton-Ouhabi a,b, ¨ a,b, Isabelle Accoceberry a,b,c,n Thierry Noel a b c
Universite´ de Bordeaux, Microbiologie Fondamentale et Pathoge´nicite´ UMR 5234, F-33000 Bordeaux, France CNRS, Microbiologie Fondamentale et Pathoge´nicite´, UMR 5234, F-33000 Bordeaux, France Centre Hospitalier Universitaire de Bordeaux, Laboratoire de Parasitologie-Mycologie, F-33000 Bordeaux, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 December 2012 Accepted 8 January 2013
We report on a fatal invasive infection due to the ascomycetous fungus Neocosmospora vasinfecta, in a 20-year-old European patient suffering from an acute lymphoblastic leukemia. The infection could not be controlled by a bitherapy combining liposomal amphotericin B and voriconazole. This is the second case of disseminated infection reported with this unusual fungus, which develops under its teleomorphic state, is fully resistant to all systemic antifungals, and which is known to live in tropical countries. & 2013 International Society for Human and Animal Mycology. Published by Elsevier B.V All rights reserved.
Keywords: Neocosmospora vasinfecta Teleomorph Antifungal-resistance Invasive fungal infection
1. Introduction Neocosmospora vasinfecta is a filamentous ascomycete belonging to the order Hypocreales known to be a plant pathogen responsible for root- and fruit-rot, and seedling damping off in a large variety of plants [1,2]. It is a common soil-borne fungus normally isolated in (sub)tropical areas, which is now considered by certain authors as a possible new emerging pathogen for humans, in spite of a very low incidence in human pathology so far [2,3]. The cases reported included a leg granuloma in a renal transplant recipient [3], a posttraumatic osteoarthritis [1] and a corneal ulcer in immunocompetent patients [4], and a fatal disseminated infection in a patient with acute nonlymphocytic leukemia [5]. We report here a case of fatal systemic N. vasinfecta infection in a patient with an acute B-lymphoblastic leukemia. This is the fifth case of human infection reported with this unusual fungal species and the second case of disseminated infection.
2. Case A 20-year-old man, native of France, was diagnosed in August 2008 with a B-cell acute lymphoblastic leukemia carrying the n Corresponding author at: Universite´ de Bordeaux, Microbiologie Fondamentale et Pathoge´nicite´ UMR 5234, F-33000 Bordeaux, France. Tel.: þ33 5 57 57 48 44; fax: þ 33 5 57 57 48 03. E-mail address: [email protected] (I. Accoceberry).
t [4,11] translocation and blasts negative for CD20 and CD10. The patient was treated according to the GRAALL 2005 protocol NCT00327678 (Group for Research in Adult Acute Lymphoblastic Leukemia). Complete remission was achieved in October 2008, but the scheduled allogenic stem cell transplantation (HSCT) could not be performed because no compatible donor had been identified. Intensification therapy was started, and the good condition of the patient allowed him back to work part-time in May 2009, and full-time in September 2009. On December 2009, a subsequent medullar relapse was diagnosed along with a H1N1 influenza pneumonia. The patient was treated with L-asparaginase encapsulated within erythrocytes (GRASPAs), according to the GRASPALL-protocol 2005-01 [6]. Bone marrow examination showed persistence of 490% blast cells. After a second-line rescue therapy, the patient received in March 2010 an allogenic stem cell transplant (Australian cord blood with two mismatches on class I, 2.7 107 total nucleated cells/kg and 0.12 106 CD34þ cells/kg). On Day 0 (D0) of HSCT, two peri-umbilical papules of 10–15 mm diameter, painful, slightly erythematous but not pruriginous were noted. Histopathological examination of a papule biopsy revealed branched, hyaline, septate hyphae invading the reticular dermis and the dermo-hypodermic junction. Intravenous bitherapy combining liposomal amphotericin B (3 mg/kg/j) and voriconazole (600 mg/kg/day for the first 48 h, then 400 mg/kg/day) was immediately started. On D1, computed tomography showed one macronodule (3 cm diameter) in the right upper lobe of lung, and no sinus abnormalities.
2211-7539/$ - see front matter & 2013 International Society for Human and Animal Mycology. Published by Elsevier B.V All rights reserved. http://dx.doi.org/10.1016/j.mmcr.2013.01.004
F. Gabriel et al. / Medical Mycology Case Reports 2 (2013) 44–47
Serum galactomannan (GM) assay (Platelia& Aspergillus Ag Kit, Bio-Rad), performed twice a week, was positive on D3 (index¼ 0.53). On D4, a high GM assay index (5.8) was measured on a bronchoalveolar lavage (BAL), whereas no Aspergillus grew from the BAL culture. Blood cultures were performed daily from D0. Two blood cultures inoculated on D7 and D8 yielded fungi, respectively after 3 days of growth on fungal media (Mycosis Bactec, Becton-Dickinson, United States), and 8 days of growth on aerobic media. It is noteworthy that, over the period from D0 to D10, 33 other aerobic and anaerobic flasks, and one Mycosis flask, remained negative. The patient received lenograstim, but remained in aplasia. In spite of the antifungal bitherapy, and of a voriconazole blood concentration of 7 mg/ml, the infection continued to disseminate rapidly. Other skin lesions appeared on abdomen, legs and skull. Myocardial injury was suspected from a T wave inversion in right precordial leads (V1–V4) along with troponin and myoglobin increased levels. The patient finally died at D12. The culture of skin biopsy yielded a mold that grew rapidly within two days on Sabouraud-glucose-agar medium containing chloramphenicol and cycloheximide at 25 1C and on Sabouraud-glucose-agar medium containing chloramphenicol without cycloheximide at 30 1C, forming white lanate colonies (Fig. 1). Microscopic examination of slide cultures showed septate hyaline hyphae, simple conidiophores, phialides, and fusoı¨d or ellipsoidal microconidia, sometimes twoseptate. Phialides were either cylindrical or aciculate, solitary or produced as a component of a complex branching system. Discrimination between Fusarium and Acremonium genus was difficult with such a microscopic morphology. After 8 days of incubation, orange to copper-colored fruiting bodies developed. The fruiting bodies were identified as perithecial ascomata and contained monoseriate ascus with 8 ascospores inside. Mature ascospores were globose to ellipsoı¨dal and possessed a rough ornamented thick wall. Molecular identification was performed by PCR amplification and nucleotide sequencing of the internal transcribed sequence (ITS) of the ribosomal RNA genes, a segment of the 18S rDNA gene, and a part of the Ef1-a translation elongation factor (TEF1) gene, as previously described [7]. Ribosomal ITS regions were amplified using the universal primer pair ITS1 (50 -TCCGTAGGTGAACCTGCGG-30 ) and ITS4 (50 -TCCTCCGCTTATTGATATGC-30 ). Region of the 18S rDNA gene was amplified using the primer pair NS3 (50 -GCAAGTCTGGTGCCAGCAGCC-30 ) and NS4 (50 -CTTCCGTCAATTCCTTTAAG-30 ). A part of the TEF1 gene was amplified using the primer pair EF1F (50 ATGGGTAAGGAGGACAAGACTC-30 ) and EF1R (50 TGGAGATACCAGCCTCGAAC-30 ) which were designed from previous work [8]. DNA was extracted from mycelia cultured in liquid YPD (yeast extract 1%, peptone 2%, glucose 2%) during 72 h at 30 1C under gentle agitation (150 rpm). Mycelial pellets were centrifuged (5000 g, 10 min, 4 1C) washed with sterile water and ground into a fine powder under liquid nitrogen. Total genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen) following the recommendations of the manufacturer. PCR products were synthesized, using HiFidelity Taq-polymerase (Qiagen), and purified with QIAquick PCR purification kit (Qiagen). ITS1, ITS4, NS3, NS4, EF1F, EF1R were used individually as primer to sequence both strands of each PCR product with ABIPrism Dye Terminator Cycle Sequencing Ready Reaction v1.1 Kit (Applied Biosystems) according to the manufacturer’s recommendations. The nucleotide sequences of ITS (GenBank accession number JX997932), and of parts of the 18S rDNA gene (GenBank accession number JX997933) and EF1a gene (GenBank accession number JX997934) were compared with those of the databases of the National Center for Biotechnology Information website (http:// www.ncbi.nlm.nih.gov) using the Basic Local Alignment Search Tool (BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequence identity (Evalue 0) was found with N. vasinfecta. Antifungal susceptibility testing
45
was performed by E-test (BioMe´rieux) on RPMI medium and revealed multiresistance. The MIC values were: amphotericin B432 mg/ml, voriconazole 24 mg/ml at 24 h then 432 mg/ml at 48 h, posaconazole 432 mg/ml, and caspofungin 432 mg/ml.
3. Discussion The genus Neocosmospora belongs to the Nectriaceae family and contains several species mainly pathogenic for plants [2]. Their occurrence in human pathology is rare. However, the species N. vasinfecta has been reported to cause a broad spectrum of infections, including superficial infections such as corneal ulcer [4], locally invasive infections, such as leg granuloma and osteoarthritis [1,3], and one disseminated infection [5]. The clinical aspects and the severity of Neocosmospora infections largely depend on the immune status of the patients [9]. Neocosmospora is a pathogen that can develop in the immunocompetent patient [1], through a direct traumatic inoculation. Some professional activities can be considered as a risk factor, like farming and gardening, as reported for the farmers growing groundnuts and cotton in India [4]. In the present clinical case, the patient with an acute B-lymphoblastic leukemia was severely immunocompromised and though very susceptible to infectious diseases. However, the isolation of N. vasinfecta was intriguing. The fungus is known to live in the soil of tropical areas and in the preceding clinical cases, the patients were originating from, or had traveled recently in, Africa [1,3,5], or South-India [4]. In the present case, the patient originated from the southwest of France, and no journey in a tropical country was documented. The critical question is how the patient was infected by the fungus. Microbial sampling in the close environment of the patient at the hospital did not reveal the presence of N. vasinfecta. The patient was pastry apprentice. One possibility is that he could have been in contact with the fungus while manipulating flours or plant and fruit products used in pastry, collected or manufactured in tropical countries and imported in Europe. As for plants, animals, virus and other microorganisms, this poses the question of the circulation of normally endemic potential pathogenic fungus around the world through human activities. Concomitantly to the HSCT needed to treat the leukemia, the patient started to develop multiple nodular skin lesions hosting filamentous elements that rapidly spread to other body sites within few days, as the consequence of an invasive, metastatic fungal infection. The cultures from the skin samples yielded a rapidly growing mycelium that had microscopic features close to Fusarium species, except that macroconidia were lacking, and also close to Acremonium species due to the presence of long aciculate phialides. Such a possible confusion was frequently reported in the literature for the genus Neocosmospora [2]. In fact, a molecular characterization of different anamorphs and teleomorphs of the Nectria/Fusarium complex revealed that the Neocosmospora genus belong to the Fusarium complex and is completely included in a Fusarium solani specific clade [10] that has the particularity to harbor all the races known to be pathogenic for both plants and animals [8,11,12]. In the present clinical case, it is also interesting to underline that serum and brochoalveolar fluid turned positive for galactomannan, whilst all cultures were negative for Aspergillus. One may conclude that the cell wall of N. vasinfecta probably contain GM antigens, as already reported for some Fusarium species [13], and that N. vasinfecta can be added to the list of non-Aspergillus fungi that may be detected by GM assay. N. vasinfecta is homothallic, that is to say that a single thallus only is required for completion of the sexual reproduction. After 10–15 days of incubation on Sabouraud agar, the mycelium turned pink to orange colored due to the production of numerous
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F. Gabriel et al. / Medical Mycology Case Reports 2 (2013) 44–47
Fig. 1. Neocosmospora vasinfecta (a) mycelium growing on Sabouraud-Chloramphenicol-cycloheximide medium after 10 days incubation at 25 1C. (b) Mycelium differentiating numerous orange perithecia on Sabouraud-Chloramphenicol-cycloheximide medium after 15 days incubation at 25 1C. (c) Hyaline hyphae with one polyphialide, and two-septate fusoı¨d conidia (1200 ). (d) Hyaline hyphae with monophialides and one-celled conidia with truncated base (600 ). (e) Hyaline hyphae with solitary or branched aciculate phialides, fusoı¨d conidia, and numerous thick-walled ascospores (400 ). (f) Isolated perithecia (100 ). (g) Periphyses constituting the neck of the opercula of the perithecium through which ascospores are released when matures (400 ). (h) Content of a young perithecium, showing cylindrical asci containing eight ascospores (100 ).
F. Gabriel et al. / Medical Mycology Case Reports 2 (2013) 44–47
perithecia containing asci and thick-walled ascospores. Interestingly, the sexual reproduction triggered spontaneously in vitro, under non-restrictive nutritional conditions, and took place concomitantly with the production of conidia. The fact that a fungus under its teleomorphic form can be responsible for an invasive infection in human can contribute to the actual debate which try to decipher the relationships between sex and pathogenicity in fungi [14,15]. For a long time, it was believed that the meiotic program of the main human fungal pathogens was switched off to the benefit of the pathogenic program. However, analyses of the data from fungal genome sequencing have shown that most pathogens have conserved genes for mating and meiosis, even the species that do not use sexual reproduction. Furthermore, it was recently discovered that some human pathogens could have conserved a cryptic sexual reproduction ability, such as Aspergillus (Neosartorya) fumigatus [16] or Aspergillus (Petromyces) flavus [17]. In the Fusarium clade, more precisely in the plant pathogen F. graminearum (teleomorph Gibberella zeae), there are now several molecular evidences that suggest biological correlation between the genes involved in sexual reproduction and the pathogenic developmental program of the fungus [18,19]. Even if the relationship between sex and virulence is more complex to demonstrate for human fungal pathogens [14], all these arguments tend to indicate that some fungal species could have retained the mating and meiotic genes not only, or not necessary, as a tool for creating genetic diversity, but also for the regulation of genes involved in pathogenesis. N. vasinfecta could be taken as an example to support this idea: its homothallism does not serve genetic diversity but contributes to homozygosity. Accordingly, one may hypothesize that the genes needed for the sexual reproduction of N. vasinfecta have been conserved during the evolution because they are important for other biological pathways, such as the pathogenic developmental stage of the fungus. Despite the association of voriconazole and liposomal amphotericin B, the patient died from the disseminated infection. In vitro, antifungal susceptibility tests revealed a high-level multiresistance to all antifungals available for systemic therapy, in particular to amphotericin B and voriconazole. It was thus conceivable that the mycological resistance was at least in part responsible for the failure of the treatment. The previous cases reporting N. vasinfecta infections also reported antifungal resistance in vitro to candins and 5-fluorocytosine. Among triazoles, resistance also prevails. MICs to voriconazole, itraconazole and posaconazole varying from 8 to 416 mg/ml were reported [8]. However, a possible susceptibility to voriconazole has already been observed for one isolate responsible for a systemic infection [5]. Likewise, even though resistance to amphotericin B has been often reported [5,8], some isolates of N. vasinfecta exhibit low MIC [4] allowing to envision the use of a liposomal amphotericin B therapy. In fact, the pattern of susceptibility and resistance to antifungals of N. vasinfecta mimics completely that of Fusarium [8], which is fully compatible with the linkage of N. vasinfecta to the clade of the Fusarium genus, and in the majority of the cases it leaves little chance of success to the antifungal therapy.
Conflict of interest The authors have no conflicts of interest. The authors are responsible for the content and writing of the paper.
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Acknowledgments The authors would like to acknowledge the Genotyping and Sequencing facility of Bordeaux for participating to the sequencing of ITS, 18S and 26S rDNA, and elongation factor-1a gene.
References [1] Kac G, Piriou P, Gueho E, Roux P, Tremoulet J, Denis M, et al. Osteoarthritis caused by Neocosmospora vasinfecta. Medical Mycology 1999;37:213–217. ¨ [2] Manikandan P, Va´gvolgyi C, Narendran V, Panneer Selvam K, Kredics L. Neocosmosporas. In: Liu D, editor. Molecular detection of human fungal pathogens. CRC Press; 2011 p. 449–58. [3] Chandenier J, Hayette MP, De Bie vre C, Westeel PF, Petit J, Achard JM, et al. Tume´faction de la jambe a Neocosmospora vasinfecta chez un transplante´ re´nal - Premier cas d’infection humaine. Journal de Mycologie Me´dicale 1993;3:165–168. [4] Manikandan P, Vismer HF, Kredics L, Do´czi I, Marasas WFO, Bhaskar M, et al. Corneal ulcer due to Neocosmospora vasinfecta in an immunocompetent patient. Medical Mycology 2007;46:279–284. ¨ H, Sohngen ¨ [5] Cornely OA, Chemnitz J, Brochhagen HG, Lemmer K, Schutt D, et al. Disseminated Neocosmospora vasinfecta infection in a patient with acute nonlymphocytic leukemia. Emerging Infectious Diseases 2001;7:149. [6] Domenech C, Thomas X, Chabaud S, Baruchel A, Gueyffier F, Mazingue F, et al. L-asparaginase loaded red blood cells in refractory or relapsing acute lymphoblastic leukaemia in children and adults: results of the GRASPALL 2005-01 randomized trial. British Journal of Haematology 2011;153:58–65. [7] Gabriel F, Noel T, Accoceberry I. Lindnera (Pichia) fabianii blood infection after mesenteric ischemia. Medical Mycology 2012;50:310–314. [8] O’Donnell K, Sutton DA, Fothergill A, McCarthy D, Rinaldi MG, Brandt ME, et al. Molecular phylogenetic diversity, multilocus haplotype nomenclature, and in vitro antifungal resistance within the Fusarium solani species complex. Journal of Clinical Microbiology 2008;46:2477–2490. [9] Nucci M, Anaissie E. Fusarium infections in immunocompromised patients. Clinical Microbiology Reviews 2007;20:695–704. [10] O’Donnell K, Sutton DA, Rinaldi MG, Magnon KC, Cox PA, Revankar SG, et al. Genetic diversity of human pathogenic members of the Fusarium oxysporum complex inferred from multilocus DNA sequence data and amplified fragment length polymorphism analyses: evidence for the recent dispersion of a geographically widespread clonal lineage and nosocomial origin. Journal of Clinical Microbiology 2004;42:5109–5120. [11] Zhang N, O’Donnell K, Sutton DA, Nalim FA, Summerbell RC, Padhye AA, et al. Members of the Fusarium solani species complex that cause infections in both humans and plants are common in the environment. Journal of Clinical Microbiology 2006;44:2186–2190. [12] Mehl HL, Epstein L. Fusarium solani species complex isolates conspecific with Fusarium solani f. sp. cucurbitae race 2 from naturally infected human and plant tissue and environmental sources are equally virulent on plants, grow at 37 1C and are interfertile. Environmental Microbiology 2007;9:2189–2199. [13] Tortorano AM, Esposto MC, Prigitano A, Grancini A, Ossi C, Cavanna C, et al. Cross-reactivity of Fusarium spp. in the Aspergillus galactomannan enzymelinked immunosorbent assay. Journal of Clinical Microbiology 2011;50: 1051–1053. [14] Butler G. Fungal sex and pathogenesis. Clinical Microbiology Reviews 2010;23:140–159. [15] Heitman J. Evolution of eukaryotic microbial pathogens via covert sexual reproduction. Cell Host & Microbe 2010;8:86–99. [16] O’Gorman CM, Fuller HT, Dyer PS. Discovery of a sexual cycle in the opportunistic fungal pathogen Aspergillus fumigatus. Nature 2008;457: 471–474. [17] Horn BW, Moore GG, Carbone I. Sexual reproduction in Aspergillus flavus. Mycologia 2009;101:423–429. [18] Jonkers W, Dong Y, Broz K, Corby Kistler H. The wor1-like protein fgp1 regulates pathogenicity, toxin synthesis and reproduction in the phytopathogenic fungus Fusarium graminearum. PLoS Pathogens 2012;8:e1002724. [19] Lee J, Myong K, Kim J-E, Kim H-K, Yun S-H, Lee Y-W. FgVelB globally regulates sexual reproduction, mycotoxin production and pathogenicity in the cereal pathogen Fusarium graminearum. Microbiology 2012;158:1723–1733.
Contents lists available at ScienceDirect
Medical Mycology Case Reports journal homepage: www.elsevier.com/locate/mmcr
A disseminated infection with the antifungal-multiresistant teleomorphic fungus Neocosmospora vasinfecta in a patient with acute B-lymphoblastic leukemia Fre´de´ric Gabriel a,b,c, Mahussi D’Almeida a,b,c, Olivier Albert a,b,c, Vale´rie Fitton-Ouhabi a,b, ¨ a,b, Isabelle Accoceberry a,b,c,n Thierry Noel a b c
Universite´ de Bordeaux, Microbiologie Fondamentale et Pathoge´nicite´ UMR 5234, F-33000 Bordeaux, France CNRS, Microbiologie Fondamentale et Pathoge´nicite´, UMR 5234, F-33000 Bordeaux, France Centre Hospitalier Universitaire de Bordeaux, Laboratoire de Parasitologie-Mycologie, F-33000 Bordeaux, France
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 December 2012 Accepted 8 January 2013
We report on a fatal invasive infection due to the ascomycetous fungus Neocosmospora vasinfecta, in a 20-year-old European patient suffering from an acute lymphoblastic leukemia. The infection could not be controlled by a bitherapy combining liposomal amphotericin B and voriconazole. This is the second case of disseminated infection reported with this unusual fungus, which develops under its teleomorphic state, is fully resistant to all systemic antifungals, and which is known to live in tropical countries. & 2013 International Society for Human and Animal Mycology. Published by Elsevier B.V All rights reserved.
Keywords: Neocosmospora vasinfecta Teleomorph Antifungal-resistance Invasive fungal infection
1. Introduction Neocosmospora vasinfecta is a filamentous ascomycete belonging to the order Hypocreales known to be a plant pathogen responsible for root- and fruit-rot, and seedling damping off in a large variety of plants [1,2]. It is a common soil-borne fungus normally isolated in (sub)tropical areas, which is now considered by certain authors as a possible new emerging pathogen for humans, in spite of a very low incidence in human pathology so far [2,3]. The cases reported included a leg granuloma in a renal transplant recipient [3], a posttraumatic osteoarthritis [1] and a corneal ulcer in immunocompetent patients [4], and a fatal disseminated infection in a patient with acute nonlymphocytic leukemia [5]. We report here a case of fatal systemic N. vasinfecta infection in a patient with an acute B-lymphoblastic leukemia. This is the fifth case of human infection reported with this unusual fungal species and the second case of disseminated infection.
2. Case A 20-year-old man, native of France, was diagnosed in August 2008 with a B-cell acute lymphoblastic leukemia carrying the n Corresponding author at: Universite´ de Bordeaux, Microbiologie Fondamentale et Pathoge´nicite´ UMR 5234, F-33000 Bordeaux, France. Tel.: þ33 5 57 57 48 44; fax: þ 33 5 57 57 48 03. E-mail address: [email protected] (I. Accoceberry).
t [4,11] translocation and blasts negative for CD20 and CD10. The patient was treated according to the GRAALL 2005 protocol NCT00327678 (Group for Research in Adult Acute Lymphoblastic Leukemia). Complete remission was achieved in October 2008, but the scheduled allogenic stem cell transplantation (HSCT) could not be performed because no compatible donor had been identified. Intensification therapy was started, and the good condition of the patient allowed him back to work part-time in May 2009, and full-time in September 2009. On December 2009, a subsequent medullar relapse was diagnosed along with a H1N1 influenza pneumonia. The patient was treated with L-asparaginase encapsulated within erythrocytes (GRASPAs), according to the GRASPALL-protocol 2005-01 [6]. Bone marrow examination showed persistence of 490% blast cells. After a second-line rescue therapy, the patient received in March 2010 an allogenic stem cell transplant (Australian cord blood with two mismatches on class I, 2.7 107 total nucleated cells/kg and 0.12 106 CD34þ cells/kg). On Day 0 (D0) of HSCT, two peri-umbilical papules of 10–15 mm diameter, painful, slightly erythematous but not pruriginous were noted. Histopathological examination of a papule biopsy revealed branched, hyaline, septate hyphae invading the reticular dermis and the dermo-hypodermic junction. Intravenous bitherapy combining liposomal amphotericin B (3 mg/kg/j) and voriconazole (600 mg/kg/day for the first 48 h, then 400 mg/kg/day) was immediately started. On D1, computed tomography showed one macronodule (3 cm diameter) in the right upper lobe of lung, and no sinus abnormalities.
2211-7539/$ - see front matter & 2013 International Society for Human and Animal Mycology. Published by Elsevier B.V All rights reserved. http://dx.doi.org/10.1016/j.mmcr.2013.01.004
F. Gabriel et al. / Medical Mycology Case Reports 2 (2013) 44–47
Serum galactomannan (GM) assay (Platelia& Aspergillus Ag Kit, Bio-Rad), performed twice a week, was positive on D3 (index¼ 0.53). On D4, a high GM assay index (5.8) was measured on a bronchoalveolar lavage (BAL), whereas no Aspergillus grew from the BAL culture. Blood cultures were performed daily from D0. Two blood cultures inoculated on D7 and D8 yielded fungi, respectively after 3 days of growth on fungal media (Mycosis Bactec, Becton-Dickinson, United States), and 8 days of growth on aerobic media. It is noteworthy that, over the period from D0 to D10, 33 other aerobic and anaerobic flasks, and one Mycosis flask, remained negative. The patient received lenograstim, but remained in aplasia. In spite of the antifungal bitherapy, and of a voriconazole blood concentration of 7 mg/ml, the infection continued to disseminate rapidly. Other skin lesions appeared on abdomen, legs and skull. Myocardial injury was suspected from a T wave inversion in right precordial leads (V1–V4) along with troponin and myoglobin increased levels. The patient finally died at D12. The culture of skin biopsy yielded a mold that grew rapidly within two days on Sabouraud-glucose-agar medium containing chloramphenicol and cycloheximide at 25 1C and on Sabouraud-glucose-agar medium containing chloramphenicol without cycloheximide at 30 1C, forming white lanate colonies (Fig. 1). Microscopic examination of slide cultures showed septate hyaline hyphae, simple conidiophores, phialides, and fusoı¨d or ellipsoidal microconidia, sometimes twoseptate. Phialides were either cylindrical or aciculate, solitary or produced as a component of a complex branching system. Discrimination between Fusarium and Acremonium genus was difficult with such a microscopic morphology. After 8 days of incubation, orange to copper-colored fruiting bodies developed. The fruiting bodies were identified as perithecial ascomata and contained monoseriate ascus with 8 ascospores inside. Mature ascospores were globose to ellipsoı¨dal and possessed a rough ornamented thick wall. Molecular identification was performed by PCR amplification and nucleotide sequencing of the internal transcribed sequence (ITS) of the ribosomal RNA genes, a segment of the 18S rDNA gene, and a part of the Ef1-a translation elongation factor (TEF1) gene, as previously described [7]. Ribosomal ITS regions were amplified using the universal primer pair ITS1 (50 -TCCGTAGGTGAACCTGCGG-30 ) and ITS4 (50 -TCCTCCGCTTATTGATATGC-30 ). Region of the 18S rDNA gene was amplified using the primer pair NS3 (50 -GCAAGTCTGGTGCCAGCAGCC-30 ) and NS4 (50 -CTTCCGTCAATTCCTTTAAG-30 ). A part of the TEF1 gene was amplified using the primer pair EF1F (50 ATGGGTAAGGAGGACAAGACTC-30 ) and EF1R (50 TGGAGATACCAGCCTCGAAC-30 ) which were designed from previous work [8]. DNA was extracted from mycelia cultured in liquid YPD (yeast extract 1%, peptone 2%, glucose 2%) during 72 h at 30 1C under gentle agitation (150 rpm). Mycelial pellets were centrifuged (5000 g, 10 min, 4 1C) washed with sterile water and ground into a fine powder under liquid nitrogen. Total genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen) following the recommendations of the manufacturer. PCR products were synthesized, using HiFidelity Taq-polymerase (Qiagen), and purified with QIAquick PCR purification kit (Qiagen). ITS1, ITS4, NS3, NS4, EF1F, EF1R were used individually as primer to sequence both strands of each PCR product with ABIPrism Dye Terminator Cycle Sequencing Ready Reaction v1.1 Kit (Applied Biosystems) according to the manufacturer’s recommendations. The nucleotide sequences of ITS (GenBank accession number JX997932), and of parts of the 18S rDNA gene (GenBank accession number JX997933) and EF1a gene (GenBank accession number JX997934) were compared with those of the databases of the National Center for Biotechnology Information website (http:// www.ncbi.nlm.nih.gov) using the Basic Local Alignment Search Tool (BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequence identity (Evalue 0) was found with N. vasinfecta. Antifungal susceptibility testing
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was performed by E-test (BioMe´rieux) on RPMI medium and revealed multiresistance. The MIC values were: amphotericin B432 mg/ml, voriconazole 24 mg/ml at 24 h then 432 mg/ml at 48 h, posaconazole 432 mg/ml, and caspofungin 432 mg/ml.
3. Discussion The genus Neocosmospora belongs to the Nectriaceae family and contains several species mainly pathogenic for plants [2]. Their occurrence in human pathology is rare. However, the species N. vasinfecta has been reported to cause a broad spectrum of infections, including superficial infections such as corneal ulcer [4], locally invasive infections, such as leg granuloma and osteoarthritis [1,3], and one disseminated infection [5]. The clinical aspects and the severity of Neocosmospora infections largely depend on the immune status of the patients [9]. Neocosmospora is a pathogen that can develop in the immunocompetent patient [1], through a direct traumatic inoculation. Some professional activities can be considered as a risk factor, like farming and gardening, as reported for the farmers growing groundnuts and cotton in India [4]. In the present clinical case, the patient with an acute B-lymphoblastic leukemia was severely immunocompromised and though very susceptible to infectious diseases. However, the isolation of N. vasinfecta was intriguing. The fungus is known to live in the soil of tropical areas and in the preceding clinical cases, the patients were originating from, or had traveled recently in, Africa [1,3,5], or South-India [4]. In the present case, the patient originated from the southwest of France, and no journey in a tropical country was documented. The critical question is how the patient was infected by the fungus. Microbial sampling in the close environment of the patient at the hospital did not reveal the presence of N. vasinfecta. The patient was pastry apprentice. One possibility is that he could have been in contact with the fungus while manipulating flours or plant and fruit products used in pastry, collected or manufactured in tropical countries and imported in Europe. As for plants, animals, virus and other microorganisms, this poses the question of the circulation of normally endemic potential pathogenic fungus around the world through human activities. Concomitantly to the HSCT needed to treat the leukemia, the patient started to develop multiple nodular skin lesions hosting filamentous elements that rapidly spread to other body sites within few days, as the consequence of an invasive, metastatic fungal infection. The cultures from the skin samples yielded a rapidly growing mycelium that had microscopic features close to Fusarium species, except that macroconidia were lacking, and also close to Acremonium species due to the presence of long aciculate phialides. Such a possible confusion was frequently reported in the literature for the genus Neocosmospora [2]. In fact, a molecular characterization of different anamorphs and teleomorphs of the Nectria/Fusarium complex revealed that the Neocosmospora genus belong to the Fusarium complex and is completely included in a Fusarium solani specific clade [10] that has the particularity to harbor all the races known to be pathogenic for both plants and animals [8,11,12]. In the present clinical case, it is also interesting to underline that serum and brochoalveolar fluid turned positive for galactomannan, whilst all cultures were negative for Aspergillus. One may conclude that the cell wall of N. vasinfecta probably contain GM antigens, as already reported for some Fusarium species [13], and that N. vasinfecta can be added to the list of non-Aspergillus fungi that may be detected by GM assay. N. vasinfecta is homothallic, that is to say that a single thallus only is required for completion of the sexual reproduction. After 10–15 days of incubation on Sabouraud agar, the mycelium turned pink to orange colored due to the production of numerous
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Fig. 1. Neocosmospora vasinfecta (a) mycelium growing on Sabouraud-Chloramphenicol-cycloheximide medium after 10 days incubation at 25 1C. (b) Mycelium differentiating numerous orange perithecia on Sabouraud-Chloramphenicol-cycloheximide medium after 15 days incubation at 25 1C. (c) Hyaline hyphae with one polyphialide, and two-septate fusoı¨d conidia (1200 ). (d) Hyaline hyphae with monophialides and one-celled conidia with truncated base (600 ). (e) Hyaline hyphae with solitary or branched aciculate phialides, fusoı¨d conidia, and numerous thick-walled ascospores (400 ). (f) Isolated perithecia (100 ). (g) Periphyses constituting the neck of the opercula of the perithecium through which ascospores are released when matures (400 ). (h) Content of a young perithecium, showing cylindrical asci containing eight ascospores (100 ).
F. Gabriel et al. / Medical Mycology Case Reports 2 (2013) 44–47
perithecia containing asci and thick-walled ascospores. Interestingly, the sexual reproduction triggered spontaneously in vitro, under non-restrictive nutritional conditions, and took place concomitantly with the production of conidia. The fact that a fungus under its teleomorphic form can be responsible for an invasive infection in human can contribute to the actual debate which try to decipher the relationships between sex and pathogenicity in fungi [14,15]. For a long time, it was believed that the meiotic program of the main human fungal pathogens was switched off to the benefit of the pathogenic program. However, analyses of the data from fungal genome sequencing have shown that most pathogens have conserved genes for mating and meiosis, even the species that do not use sexual reproduction. Furthermore, it was recently discovered that some human pathogens could have conserved a cryptic sexual reproduction ability, such as Aspergillus (Neosartorya) fumigatus [16] or Aspergillus (Petromyces) flavus [17]. In the Fusarium clade, more precisely in the plant pathogen F. graminearum (teleomorph Gibberella zeae), there are now several molecular evidences that suggest biological correlation between the genes involved in sexual reproduction and the pathogenic developmental program of the fungus [18,19]. Even if the relationship between sex and virulence is more complex to demonstrate for human fungal pathogens [14], all these arguments tend to indicate that some fungal species could have retained the mating and meiotic genes not only, or not necessary, as a tool for creating genetic diversity, but also for the regulation of genes involved in pathogenesis. N. vasinfecta could be taken as an example to support this idea: its homothallism does not serve genetic diversity but contributes to homozygosity. Accordingly, one may hypothesize that the genes needed for the sexual reproduction of N. vasinfecta have been conserved during the evolution because they are important for other biological pathways, such as the pathogenic developmental stage of the fungus. Despite the association of voriconazole and liposomal amphotericin B, the patient died from the disseminated infection. In vitro, antifungal susceptibility tests revealed a high-level multiresistance to all antifungals available for systemic therapy, in particular to amphotericin B and voriconazole. It was thus conceivable that the mycological resistance was at least in part responsible for the failure of the treatment. The previous cases reporting N. vasinfecta infections also reported antifungal resistance in vitro to candins and 5-fluorocytosine. Among triazoles, resistance also prevails. MICs to voriconazole, itraconazole and posaconazole varying from 8 to 416 mg/ml were reported [8]. However, a possible susceptibility to voriconazole has already been observed for one isolate responsible for a systemic infection [5]. Likewise, even though resistance to amphotericin B has been often reported [5,8], some isolates of N. vasinfecta exhibit low MIC [4] allowing to envision the use of a liposomal amphotericin B therapy. In fact, the pattern of susceptibility and resistance to antifungals of N. vasinfecta mimics completely that of Fusarium [8], which is fully compatible with the linkage of N. vasinfecta to the clade of the Fusarium genus, and in the majority of the cases it leaves little chance of success to the antifungal therapy.
Conflict of interest The authors have no conflicts of interest. The authors are responsible for the content and writing of the paper.
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Acknowledgments The authors would like to acknowledge the Genotyping and Sequencing facility of Bordeaux for participating to the sequencing of ITS, 18S and 26S rDNA, and elongation factor-1a gene.
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