MEDICINE

Nigrospora oryzae Pulmonary Infection in a Bronchogenic Cancer:an Opportunistic Invader?

Hari Pankaj Vanam1& Ranganath Deshpande2

& Krishnaveni Nayanagari3 & Vijay Sreedhar V4&

Shivaprakash Mandya Rudramurthy5

Accepted: 28 May 2020# Springer Nature Switzerland AG 2020

AbstractThe genus Nigrospora is a cosmopolitan dematiaceous ascomycetes fungus which inhabits the soil and includes both entomo-pathogenic and phytopathogenic properties. Among the evolving species of Nigrospora, the frequently isolated are Nigrosporasphaerica (N. sphaerica) and Nigrospora oryzae (N. oryzae). N. sphaerica has been implicated in human infections like cornealulcer, onychomycosis, respiratory allergies, deep mycoses, and skin infections in both immunocompetent and immunocompro-mised individuals, whereas N. oryzae is an established plant pathogen and the only reported human association is its isolationfrom human superficial skin scrapings. Here, we report a case of pulmonary infection by N. oryzae in a 55-year-old male with aneoplasm of squamous cell origin and diffuse skeletal metastasis. Bronchoalveolar lavage and biopsies of lung tissue demon-strated branching septate hyphae and extensive tumor necrosis and malignant cells, respectively. Fungal culture and molecularsequencing established N. oryzae as the etiological agent. The patient was treated with itraconazole and palliative radiotherapy.Due to the poor compliance and unfavorable effects of treatments, the patient opted for alternative therapies and succumbed dueto ongoing invasive pathogenesis. To the best of our knowledge, this is the first communication describing a dematiaceous moldN. oryzae causing opportunistic pulmonary infection in a lung neoplasm along with antifungal susceptibility data against sevenantifungal drugs using the reference CLSI method.

Keywords ITS sequencing . Phytopathogenic fungi . Opportunistic mycoses . Nigrospora oryzae . Phylogenetic analyses .

Phaeohyphomycosis . CLSIM38

Introduction

Opportunistic fungal infections by non-Aspergillus filamen-tous fungi including dematiaceous fungi have been increas-ingly reported [1]. Slowly progressive fungal disease causedby a heterogeneous group of dematiaceous fungi which arep r inc ipa l l y sap rob i c i s co l l e c t i ve ly t e rmed asphaeohyphomycosis [2]. Traumatic inoculation of the skinand the respiratory route are the important routes of transmis-sion of infection by dematiaceous fungi in immunocompro-mised (IC) hosts [3]. Human infections with Nigrospora spe-cies, especially Nigrospora sphaerica (N. sphaerica), thoughrare, were previously reported to cause corneal ulcers,onychomycosis, respiratory allergies, deep fungal infections,and skin infections in both immunocompetent and IC individ-uals [4–7]. Nigrospora oryzae (N. oryzae) is an establishedplant pathogen causing plant diseases like stem blight inIndianmustard plantations and is actively evaluated for its roleas an antimicrobial and bio-control property [8, 9]. In a survey

This article is part of the Topical Collection onMedicine

Repos i tory ITS Sequence GenBank Access ion numberMK453286.1https://www.ncbi.nlm.nih.gov/nuccore/MK453286

* Hari Pankaj Vanampankajgenome@gmail.com

1 Mycology Division, Department of Microbiology, Bhaskar MedicalCollege and General Hospital, Bhaskar Nagar, Yenkapally,Moinabad, R.R. District, Hyderabad, Telangana 500 075, India

2 Department of Pulmonology, Bhaskar Medical College and GeneralHospital, Bhaskar Nagar, Yenkapally, Moinabad, R.R. District,Hyderabad, Telangana 500 075, India

3 Department of Radiodiagnosis, Bhaskar Medical College andGeneral Hospital, Bhaskar Nagar, Yenkapally, Moinabad, R.R.District, Hyderabad, Telangana 500 075, India

4 Department of Pathology, Bhaskar Medical College and GeneralHospital, Bhaskar Nagar, Yenkapally, Moinabad, R.R. District,Hyderabad, Telangana 500 075, India

5 Department of Medical Microbiology, Postgraduate Institute ofMedical Education and Research, Chandigarh 160012, India

https://doi.org/10.1007/s42399-020-00340-x

/ Published online: 14 June 2020

SN Comprehensive Clinical Medicine (2020) 2:919–927

of dematiaceous fungi fromMalaysia, N. oryzae was reportedas an opportunistic species isolated from four-superficial skinscrapings and has never been encountered as a pulmonarypathogen [10].

H e r e , w e r e p o r t a r a r e c a s e o f p u lmon a r yphaeohyphomycosis in a 55-year-old male Indian farmer withan underlying non-small cell lung cancer (NSCLC) of squa-mous cell origin with a TNM Staging T4N3 M1c. To the bestof our knowledge, this is the first communication describing adematiaceous mold N. oryzae causing opportunistic pulmo-nary infection in a lung neoplasm along with antifungal sus-ceptibility data against seven-antifungal drugs using the refer-ence CLSI method [11].

Case Report

A 55-year-old male farmer presented to the pulmonology out-patient department (OPD) with complaints of persistent coughwith hemoptysis and fever for a duration of 2 months. Therewas no significant respiratory illness in the past and the patientwas non-diabetic and non-alcoholic. He was a smoker and aknown hypertensive on medication for 5 years. Basic investi-gations revealed normal white cell count and platelet count.Hemoglobin was 12 g/dL. On clinical examination, reducedbreath sounds in the right infra-scapular and infra-axillaryareas with stable vitals were appreciated.

On day one of the hospital stay, chest radiography revealeda large mass in the lower lobe of the right lung. A contrast-enhanced computed tomography (CECT) of axial/coronal/sagittal planes of the chest showed evidence of 85 × 62 ×52 mm solid mass lesion in the right lower lobe (LL) abuttingmajor fissure extending intra-bronchially into distal part of theright main bronchus through LL bronchi with surroundingspiculations (Fig. 1A). Furthermore, a mild central enhance-ment with a difference of 20 Hounsfield units (HU) and withan increased peripheral enhancement was noted (Fig. 1B). Noevidence of calcification or air bronchogram or any other mass

lesions in the rest of the lung parenchyma was noted.Evidence of few posterior mediastinal lymph nodesmeasuringa maximum of 11 mm on the ipsilateral side was appreciated.Evidence of small lytic lesion areas of dorsal spine was noted(Fig. 1C), all of these features were suggestive of bronchogen-ic carcinoma with a probable metastasis and hence recom-mended for a biopsy.

On hospital day 4, a bronchoscopy procedure was per-formed which showed a granulomatous necrotic tissue in theright LL bronchus with friable plaques and a mass occludingthe bronchus not allowing the scope to pass further.Bronchoalveolar lavage (BAL) and biopsy from theendobronchial tissue were taken and sent for laboratory inves-tigations. In view of high index of suspicion of carcinoma, thecase was referred to the Regional Cancer Centre for furtherevaluation and management.

Microbiological and Histopathological Investigations

The BAL specimen was subjected to 20% potassium hydrox-ide (KOH) mount, Gram’s stain, and Ziehl–Neelsen (ZN)staining and was further processed for bacterial cultures byinoculating on blood agar (BA), Lowenstein Jensen (LJ) me-dium which was incubated at 37 °C for 1–8 weeks. Fungalcultures were performed by inoculating in two tubes ofSabouraud’s dextrose agar (SDA) with 0.005 g chloramphen-icol (HiMedia, India), one incubated in ambient air at 28 °C ina BOD (Biological Oxygen Demand/Biochemical OxygenDemand (BOD)) incubator and the other at 37 °C, respective-ly. The soft tissue in multiple minute bits measuring 0.4 cm ofgrayish-white to light brown color biopsy material from theendobronchial tissue was sent for histopathological staining.

Direct Microscopy and Histopathological Examination

KOH mount of the BAL revealed plenty of sub-hyaline, sep-tate branching hyphae suggesting a fungal etiology (Fig. 2A).

Fig. 1 AAxial CECT view of mediastinal window showing a solid masslesion in the right lower lobe (LL) with surrounding spiculations (arrow-head). B Coronal CECT view showing the mass lesion infiltrating intra-

bronchially into distal part of the right main bronchus through LL bronchi(arrowhead). C Sagittal view showing small lytic areas in the dorsalvertebral spine (arrowhead)

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No bacteria were seen on a Gram’s stain and no acid-fastbacilli (AFB) seen on ZN staining. Histopathological stainingusing Hematoxylin and Eosin (H & E) stain demonstratednumerous branching septate hyphae with necrotic material inthe tissue studied (Fig. 2B). On day 5, the patient was startedwith itraconazole 200 mg/BD along with intravenous (IV)augmentin, metronidazole, and gentamycin. On hospital day7, mycelial colonies on SDA were visible on both 28 °C and37 °C temperatures, the later showing restricted growth. Nobacterial growth on BA after 1 week of incubation and noAFB on LJ medium on further incubation for 8 weeks, respec-tively. On the 15th day of hospital stay, the patient was re-ferred to the Regional Cancer Center for a CT-guided biopsyof the right lung mass which revealed findings suggestive of asquamous-originated NSCLC. Based on histopathologicaland fungal culture findings, a diagnosis of bronchogenic car-cinoma with invasive fungal infection was established and thepatient was started on palliative care with the continuation ofantifungal treatment.

Morphological Description and PhenotypicIdentification of N. oryzae

The colonies on SDA with chloramphenicol after 10 daysshowed grayish lanose surface growth with buff reverse(Fig. 3A, B). A preliminary teasing and adhesive tape impres-sion mounts with lactophenol cotton blue (LPCB) stain re-vealed a dematiaceous mold with a characteristic monoblasticconidia. A micro-slide culture on potato dextrose agar (PDA)for morphological identification showed abundant superficialbranched septate hyaline hyphae, giving rise to conidiophoreswhich are sub-hyaline to dematiaceous towards theconidiogenous cells. Conidiogenous cells were arranged lat-eral or terminal with swollen ampulliform structures givingrise to solitary monoblastic conidia at the attenuated apex.

Conidia are black, shiny, smooth, and spherical to sub-spherical which are abundantly produced. With the followingmorphological features, the isolate was identified asNigrospora spp. (Fig. 3C, D). Due to rare involvement ofthe genera of Nigrospora in pulmonary infection and its exis-tence of multiple cryptic species and potential antifungal re-sistance, the isolate was submitted in the National CultureCollection of Pathogenic Fungi (NCCPF), PostgraduateInstitute of Medical Education and Research (PGIMER),Chandigarh, India (submission no. IL_3541 (Myc-405) formolecular confirmation and AFST data.

Confirmation of N. oryzae by Molecular Identificationand Phylogenetic Analysis

Molecular identification of the culture was performed at theNCCPF, PGIMER, Chandigarh, India. The isolate was har-vested on SDA slopes for 3–4 days. The DNA extraction wascarried out using phenol:chloroform:isoamyl alcohol(25:24:1) and ethanol precipitation method. The amplificationof internal transcribed spacer regions 1 and 2 (ITS1 and ITS2)was performed in the PCR assay using the universal primerpair. The purified product of ITS PCR was processed for ITSsequencing of ribosomal DNA. Sequencing and analysis wereperformed in automated DNA sequencer-ABI Prism 3130XLGenetic Analyzer (Applied Biosystems) using ABI PRISMBigDye Terminator Cycle Sequencing Ready Reaction Kit(Applied Biosystems, Foster City, CA, USA).

The ITS sequence from the present case was comparedwith 15 other ITS sequences of type strains retrieved fromthe NCBI GenBank ITS database (https://blast.ncbi.nlm.nih.gov/Blast.cgi), ITS database from Centraal Bureau voor deStatistiek (CBS), Utrecht, The Netherlands (“www.westerdijkinstitute.nl/medical/”), respectively (Table 1).Multiple sequence comparison by log-expectation (MUSLE)

Fig. 2 Direct microscopy of BALfluid and endobronchial tissue. AExtensive retractile, branchingseptate hyphae on KOH mount(40× magnification, zoomed 2×)(arrowheads). BHistopathological stain using H&E of endobronchial tissueshowing numerous branchinghyphae and necrotic tissuereaction (40× magnification)(arrowhead)

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program of Molecular Evolutionary Genetics Analyses(MEGA7) software was used for alignment of multiple ITSsequences [12, 13]. Evolutionary and phylogenetic analysesby Maximum Likelihood (ML) method were conducted usingMEGA7 [13]. The dataset comprised a total of 16 ITS se-quences retrieved which include 11 different species ofNigrospora and rooted with outgroups Seiridium spyridicolaand Arthrinium malaysianum (Fig. 4). Phylogenetic recon-struction positioned the present ITS sequence of N. oryzaeVanam et al. closely with all the type strains ITS material ofN. oryzae along with Nigrospora gorlenkoana in the phylo-genetic tree (Fig. 4). The phylogenetic tree and evolutionaryanalyses confirmed the isolate asN. oryzae. The sequence wasdeposited in GenBank ITS databases and published in theNCBI database on 29/01/2019 with accession numberMK453286.1 and name N. oryzae strain IL3541_Myc_405.

In Vitro Antifungal Susceptibility Testing (AFST) ofN. oryzae

In vitro AFST of N. oryzae was performed against seven an-tifungal drugs using the reference broth microdilution (BMD)method as per Clinical Laboratory Standards International(CLSI) M38 third edition protocol with slight modification[11]. All antifungal (AF) powders were of reagent grade(Sigma-Aldrich, Bengaluru, India) and dissolved in dimethylsulfoxide (DMSO) except for caspofungin, which was dis-solved in sterile distilled water. The inoculum was prepared

from a 7-day-old culture on PDA incubated at 28 °C. Sporeswere harvested using sterile normal saline (0.85%) and bygently probing the tip of a transfer pipette to obtain the mixtureof conidia and hyphal fragments in a sterile tube. The conidialsuspension was adjusted to an OD of 0.20 at 530 nm.Inoculum dilutions of 1:50 were prepared using RPMI 1640media (HiMedia, India) and inoculated onto 96-well microti-ter plate containing AF drugs. Candida krusei ATCC 6258,Aspergillus flavus ATCC 20430, and Candida parapsilosisATCC 22019 were included as the quality control strains.The results of AFST revealed lower minimum inhibitory con-centrations (MIC) on voriconazole (VOR), amphotericin B(AmB), posaconazole (POS), i traconazole (ITR),anidulafungin (ANI), and higher MICs on caspofungin(CAS) and fluconazole (FLU), respectively (Table 2).

Follow-up and Outcome

On the 35th day of the course of illness, the patientcomplained of non-radiating chest pain on the right side whichaggravated coughing and right-sided abdomen pain with amoderate intensity of back pain. An investigation for a possi-ble metastasis of lung cancer to liver and adrenals was carriedout by the Regional Cancer Centre by an ultrasound-guidedFNAC (fine-needle aspiration cytology) of the liver lesionwhich revealed moderately acellular smear showing tiny clus-ters of large round to polygonal cells with hyperchromaticnucleus with moderate amount of cytoplasm admixed with

Fig. 3 Phenotypic and microscopic attributes of N. oryzae.A–BObverseand reverse of 10-day-old culture on SDA. C LPCB mount of slideculture showing conidiophores and conidiogenous cells giving rise to

abundant conidia seen in 40× magnification. D Conidia are globose orsub-globose, black, shiny, smooth, aseptate measuring 12–14 μm bornesolitarily on the dematiaceous conidiogenous cells

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inflammatory cells, fibrin strands, histiocytes in a hemorrhag-ic background with an impression of features suggestive ofmetastatic deposits favoring squamous origin.

On the 40th day, functional evaluation of the risk of skel-etal metastasis was performed using Tc-99m MDP whole-body bone scintigraphy. Findings were suggestive of carcino-ma of lung stage-IV with diffuse skeletal metastasis involvingthe right hemithorax with likely pleural involvement. Thisprompted the approval of palliative external beam radiationtherapy with 3000 centigray (cGy) given in ten fractions of300 cGy for 10 days. Due to the traditional beliefs and low-socioeconomic background, the patient was opined by the kinabout taking alternative therapies which also led to the non-compliance of the prescribed dose of palliative radiation andantifungal therapies. Amidst the skeletal metastasis and sever-ity of the disease, the patient opted for alternative therapy witha plant-based bark extract and succumbed at the end of the 3-month ordeal of cancer therapy.

Discussion

Fungal infections in patients with neoplasm can be manifestedin a protean manner and sometimes result in life-threateningevents. There are increased reports of infections caused bynon-Aspergillus filamentous molds like Mucorales,Fusarium and Scedosporium species along with a variety ofphaeoid fungi collectively where the later are termed asphaeohyphomycosis [1]. Phaeohyphomycosis in IC cancerpatients is frequently reported by Bipolaris spicifera,Cladophialophora bantiana, Ramichloridium mackenziei,and Wangiella dermatitidis [14]. Furthermore, Exserohilumrostratum was implicated in a multi-state outbreak due to po-tentially contaminated methylprednisolone acetate injections[14, 15]. Inhalation of saprobic fungal spores is almost uni-versal and a host with specific IC state like carcinoma of lungare extremely susceptible and the risk factors like chronicobstructive pulmonary disease (COPD) and liver failure may

Table 1 List of reference ITSsequences from culture collectionstrains retrieved from the NCBI,Genbank, and CBS,The Netherlands with accessionnumbers included in thephylogenetic study

Species Country origin Accessionnumbers

Source of ITS data

N. oryzae Hyderabad, India MK453286.1 NCBI, GenBank ITSdatabase

N. oryzae Rio Claro, Sao Paulo,Brazil

KR093878.1 NCBI, GenBank ITSdatabase

N. oryzae Zhengjiang, China KT224879.1 CBS and GenBank ITSdatabase

N. oryzae Puerto Rico, USA KT966519.1 NCBI, GenBank ITSdatabase

N. sphaerica Shanghai,China JN198501.1 NCBI, GenBank ITSdatabase

N. osmanthi Beijing, China NR_153474.1 NCBI, GenBank ITSdatabase

N. gorlenkoana CBS Beijing, China NR_153476.1 NCBI, GenBank ITSdatabase

N. hainanensis Beijing, China KX986091.1 NCBI, GenBank ITSdatabase

N. guilinensis Beijing, China NR_153472.1 NCBI, GenBank ITSdatabase

N. pyriformis Beijing, China NR_153469.1 NCBI, GenBank ITSdatabase

N. musae CBS Beijing, China NR_153478.1 NCBI, GenBank ITSdatabase

N. lacticolonia Beijing, China NR_153471.1 NCBI, GenBank ITSdatabase

N. rubi Beijing, China NR_153470.1 NCBI, GenBank ITSdatabase

N. zimmermanii CBS Beijing, China NR_153485.1 NCBI, GenBank ITSdatabase

Seiridium spyridicola CBS Uppsalalaan,The Netherlands

NR_160578.1 NCBI, GenBank ITSdatabase

Arthrinium malaysianumCBS

Uppsalalaan,The Netherlands

NR_120273.1 NCBI, GenBank ITSdatabase

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result in poor prognoses. The environmental factors in a trop-ical country like India along with farming, smoking, living inthe countryside, and variation in the weather increase the riskmanifolds. The prognostic factors that determine the outcomeof infections by non-Aspergillus molds do not differ frompatients with invasive aspergillosis (IA). A recent review con-cludes that the role of uncontrolled malignancy, disseminateddisease, and use of potent immunosuppressive agents are the

harbinger for a poor outcome of the disease [16]. The presentcase is a reflection of increased risk of environmental expo-sure and positive prognostic factors of having an uncontrolledmalignancy with rapid metastasis to liver, adrenal, andskeleton.

The clinical manifestation owing to non-Aspergillusmoldsare non-specific and timely diagnosis of etiological agent andearly initiation of the antifungal therapy are the cornerstone ofmanagement. Currently along with the conventional methods,molecular methods identifying the pathogens in tissues andsterile body fluids are increasingly evaluated for routine diag-nostic use. Diagnoses of a proven invasive fungal disease(IFD) essentially requires an inter-departmental coordination.In 2018, European Organization for Research and Treatmentof Cancer (EORTC) and Mycoses Study Group (MSG) withthe German Society for Hematology and Medical Oncology(AGIHO) amended the guidelines for diagnoses of IFD inhematology and oncology patients. The criteria include theassessment of clinical signs and symptoms, collecting the ster-ile specimen by endoscopic methods for mycological

Fig. 4 Phylogenetic tree based on internal transcribed spacer sequences(ITS) of 16 sequences using the Maximum Likelihood (ML) method.There were a total of 442 positions in the final dataset after eliminatinggaps and missing data among the sequences. The evolutionary historywas inferred by using the ML method based on the General TimeReversible five-parameter model (GTR+G+I). The tree with the highest

log likelihood (− 1430.98) is shown. The percentage of trees (bootstrap2000 replicates) in which the associated taxa clustered together is shownnext to the branches. The tree is drawn to scale, with branch lengthsmeasured in the number of substitutions per site. The tree is rooted withSeiridium spyridicola and Arthrinium malaysianum. The present speciesis highlighted in the tree as a “red button”

Table 2 Results ofin vitro AFST ofN. oryzae on seven-antifungal drugs usingCLSI M38 (2017) BMDmethod

Antifungal agent tested MIC in μg/mL

Amphotericin B 1

Voriconazole 0.12

Itraconazole 2

Posaconazole 1

Fluconazole 32

Caspofungin 8

Anidulafungin 0.5

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examination by microscopy and culture as well as non-cultural techniques (e.g., antigen and antibody detection, mo-lecular methods), imaging procedures, and cytological and/orhistological examination of biopsy material [17]. Imagingplays an essential role in diagnoses and treatment of lesions,but findings of non-Aspergillus mold infections overlap,hence imaging alone cannot determine the etiological agent.The histopathological evidence of hyphae accompanied bytissue damage from the lung biopsy established the presentcase as a proven case of IFD.

The genus Nigrospora is placed in the familyApiosporaceae of the phylum Ascomycota, which are pre-dominantly saprobic fungi causing plant diseases with afew reports of humans’ infection. Zimmerman (1902) in-troduced the generic name of Nigrospora as N. panici, anendophytic fungus from Indonesia. In 1927, Masoncircumscribed several Nigrospora species (spp.) includingN. oryzae, N. sphaerica, Nigrospora arundinacea, andNigrospora sacchari which are hyphomycetes and pro-duced monoblastic black-spores or conidia. Later specieslike Nigrospora javanica, Nigrospora gallarum, andN. gorlenkoana which share morphological features withN. panici and N. oryzae were regarded as synonymous. Atpresent , Mycobank reposi tory has 15 classif iedNigrospora spp., which still lack the familial placement.In 1998, based on conidial characteristics, Barnett &Hunter placed Nigrospora in Dematiaceae (Moniliales)and in 2008, While Kirk et al. further revised them andplaced Nigrospora and its sexual morph Khuskia in theOrder Trichosphaeriaceae [8]. This ambiguity in phylo-genetic relationships among Nigrospora is due to a higherlevel of species diversity. Recently, a large study fromChina on 165 isolates of Nigrospora spp. concluded with13 novel species by considering the multiple parameterslike multi-locus phylogenic analyses based on ITS,TEF1-α, and TUB2; morphological characters and hostassociations; and ecological data. In 2013, India first re-ported N. oryzae causing stem blight disease in Indianmustard (Brassica juncea) plants [9]. Furthermore, a sur-vey of Dematiaceous fungi from Malaysia, N. oryzae, wasisolated from a four-skin scraping specimen and was nev-er reported in causing pulmonary infections in humans[10]. The current status of infections caused byNigrospora spp. is tabulated as a global review(Table 3). The sequencing of ITS regions of rDNA hasbeen considered as the gold standard for the identificationof fungi. Major ITS database repositories such asGenBank which enable ready comparison of sequencesstill have an estimated 14–20% errors in archived se-quences which warrant multiple repositories for compari-son [18]. Our present ITS submission on nBLAST analy-ses on multiple public repositories showed similarity withN. oryzae sequences and phylogenetic analysis placed the

same in near clustering with reference ITS sequences ofN. oryzae.

The medical management of pulmonary infections owingto a variety of dematiaceous molds is complicated and optimaldrug and duration of therapy needed to be determined [10]. Ina hospital survey of dematiaceous fungi as opportunistic spe-cies, Yew et al. reported potential multidrug resistance (MDR)inN. oryzae in two of the four strains tested on eight-AF drugsincluding, ITR, VOR, POS, FLU, ANI, CAS, andKetaconazole (KET) [10]. The Epsilometer method (E-test),which is a commercial gradient diffusion method to facilitateAFST in the routine diagnostic laboratories, was employed.The CLSI M38 protocol is the reference testing method forfilamentous fungi including dematiaceous molds causing cu-taneous and invasive infections [11]. This is the first time weperformed reference BMD AFST against seven AF drugs asper CLSI method with minor modification. Due to larger sizeof conidia (> 8 μm), the OD for inoculum preparation wasadjusted to 0.20 at 530 nm [11]. VOR and ANI were thepotent in vitro AF drugs that inhibited N. oryzae, followedby POS and AmB. TheMIC against ITR was 2 μg/mL, whichis in a higher end, as MIC values > 2 μg/mL were consideredas intermediate in sensitivity for dematiaceous molds by someauthors [10]. However, these studies are purely based onMICvalues and there are no established guidelines on MICbreakpoints on various dematiaceous molds includingN. oryzae [10]. Comparison of the present AFST results (ref-erence BMD method) with that of E-test by Yew et al. [10]showed comparable results with AmB, FLU, CAS, with theformer showing susceptibility to N. oryzae and resistance tothe latter two AF drugs revealing anMDR pattern (MIC range8–32 μg/mL). In contrast to E-test results, the present isolateshowed susceptibility to ANI (0.5 μg/mL) in the BMD meth-od, which in turn is a superior in vitro AF agent in invasivemycoses. Therefore, inter- and intra-species diversity in drugsusceptibility exists among various genera of dematiaceousmolds along with N. oryzae. Further investigations are neededwith a large number of isolates to investigate potential MDRmechanisms in N. oryzae. Due to the feasibility, better tolera-bility and cost-effective nature of oral ITR, and a possibility oflong-term therapy in lieu of ongoing bronchogenic carcinoma,the patient was empirically started on ITR 400 mg/ day. ITRand its role in pulmonary aspergillosis were well documentedby Gupta et al., which concludes that ITRwas an effective andfeasible drug with good clinical and radiological responseswhen recommended as a long-term therapy [19].Furthermore, a request for AFST data of the isolate from thenational reference revealed MIC of 2 μg/mL against ITR,which justified the clinical utility of the drug in prevailingcircumstances.

The World Health Organization has stressed the need fortraditional, complementary, and alternative medicines(TCAM) in treatment of terminally ill cancer patients which

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in turn has been positively acknowledged by Indian oncolo-gists’ community [20, 21]. In a random study performed intwo cancer centers in northern India, 34.3% of cancer patientsopted for TCAM, in consequential to the poor prognosis andthe resultant rapid progression of the disease, or a delay inseeking immediate medical intervention [21]. India is a sub-continent with a wide geographic diversity with strong beliefs,therefore, it is not alarming to state that alternate therapiesinvolving TCAM will remain the choice of a last resort forthe cancer patients in developing countries for now.

Conclusion

Emerging invasive fungal infections in IC cancer patientsby rare etiologies often pose a challenge to clinicians inboth treatment and management. Early diagnosis is a cor-nerstone for early initiation of optimal therapies. Repeatedsampling by invasive procedures in a rapidly progressivedisease is always not feasible in a compromised host,therefore in any evidence of proven invasive disease, deci-sions must be taken swiftly, and any further manipulationsby invasive procedures must be prevented. Most of theempirically treated fungal infections may fail due to mod-ifying risk factors in the IC host or due to the emergence ofa rare pathogen that may be intrinsically resistant to someantifungal drugs. The traditional beliefs and practices incounties like India play a pivotal role in the treatment ofcancers and influence the patients in switching over toalternate therapies. These factors may partly predisposeto a non-compliance to recommended chemotherapy andantifungal therapies and may contribute a poor outcome,which is reflected in the present case scenario.

Acknowledgments We are indebted to Prof. Dr. Arunaloke Chakrabarti,Dr. Shamanth, andMrs. Sunita Gupta from NCCPF, Mycology Division,Department of Medical Microbiology, PGIMER, Chandigarh, India forITS sequencing and AFST. The authors also wish to acknowledge Dr.Jaya Latha, Professor of Radio-diagnoses at MNJ Regional CancerCentre, Hyderabad, Telangana, India for sharing the follow-up detailsof the patient. Lastly, the authors also acknowledge Dr. Ramesh,Professor of Pulmonary Medicine and Dr. P. R. Anuradha, Professor ofMicrobiology, Bhaskar Medical College and General Hospital,Telangana, India for extending their valuable opinion on thiscommunication.

Compliance with Ethical Standards

Conflict of Interest The authors declare that they have no conflicts ofinterest.

Ethical Approval Ethical approval was obtained by the institutional eth-ical committee of Bhaskar Medical College and General Hospital formycotic infections and all the procedures being performed were part ofroutine diagnoses and patient care and management.

Humans in Research Procedures followed were in accordance with theethical standards of the responsible committee on human experimentationwithin their institutions and/or with the Helsinki Declaration of 1975, asrevised in 1983.

Animals in Research This article does not contain any studies with an-imals performed by any of the authors.

Informed Consent Written informed consent for participation and pub-lication was obtained from the patient’s guardian.

Authorship Contributors All authors confirm in accordance with theCommittee on Publication Ethics (COPE) guidelines and InternationalCommittee of Medical Journal Editors (ICMJE) criteria. Each authorhad made a substantial contribution to the conception and design, acqui-sition of data, and/or analysis and interpretation of data. Each of theauthors participated in the drafting of the article are revising it critically

Table 3 Global review of Nigrospora human infections

Agent Site of Infection Country Age(years)

Gender Identification Treatment Reference

Nigrosporasphaerica

Right eye corneal ulcer India 45 Female Phenotypic andgenotypic (ITSrDNA)

Topical and oralVoriconazole

[4]

Nigrosporasphaerica

Right first toenail China 21 Male Phenotypic andgenotypic (ITSrDNA)

Oral Itraconazole [5]

Nigrosporasphaerica

Deep fungal infectionin HIV patient

South Africa 35 Male Phenotypic andgenotypic (ITSrDNA)

Itraconazole [7]

Nigrosporaoryzae

Skin scrapingsfrom human

MalaysiaDematiaceous fungisurvey

– – Phenotypic andgenotypic (ITSrDNA)

– [10]

Nigrosporaspp.

Left eye corneal ulcer India 60 Male Phenotypic Natamycin, Miconazoleand Ketoconazole

[6]

Nigrosporaoryzae

Pulmonary infection in lungcarcinoma patient

India 55 Male Phenotypic andgenotypic (ITSrDNA)

Itraconazole Presentcase

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for important intellectual content and have read before the approval of thefinal manuscript.

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