New Record of Bipolaris cynodontis: An Emerging Human Pathogen Causing Superficial Mycosis in North India

Abstract

Superficial mycosis, a common fungal infection affecting people worldwide are prevalent in the tropical and subtropical countries, mostly caused by the dermatophytes but nowadays, there is an increase in the incidence of these infections being caused by non-dermatophytic fungi and yeasts. Among non-dermatophytes, Bipolaris species, usually known to cause diseases in plants, have also emerged as potent human pathogens in the past years. Reports on Bipolaris species associated with clinical human skin samples are rare worldwide with no reports on B. cynodontis as a causal agent of superficial human skin mycoses from India. We report the first case of superficial mycosis caused by B. cynodontis that affected the feet of a 19-year-old female student from Jammu district, Jammu and Kashmir, India. Thus, the causal agent described in the research communication constitutes a new addition to the list of pathogenic non-dermatophytes associated with human skin.

Established Facts

• Superficial mycoses refers to the fungal infections restricted to the skin and its appendages.

• Dermatophytes are mainly responsible for most of the cases of superficial mycoses.

• The genus Bipolaris includes various significant phytopathogenic species (mostly affecting Poaceae).

Novel Insights

• This is the first case report of superficial human skin mycosis caused by Bipolaris cynodontis from Jammu and Kashmir, India.

• The isolated non-dermatophyte fungus is capable of causing infection in immunocompetent individuals.

Introduction

Superficial mycoses refers to the fungal infections restricted to the outermost layers of the skin and its appendages, which are caused by different species of dermatophytes, non-dermatophyte molds, yeasts, and yeast-like fungi [1]. Such infections occur worldwide with a prevalence rate of 20–25% and are continuously increasing [2]. Bipolaris is a large genus that includes >100 species [3]. This genus is worldwide in distribution with most of the species growing in soil as saprophytes and as phytopathogens, while some species, such as B. spicifera, B. australiensis, and B. hawaiiensis have been found to be capable of causing infection in both immunocompetent and immunocompromised humans [3, 4]. The genus Bipolaris include various significant phytopathogenic species mostly affecting family Poaceae, such as B. arizonica, B. bicolor, B. clavata, B. cookie, and B. oryzae [3, 5]. So far, as the association of Bipolaris with humans is concerned, a few species have been found to act as occasional opportunistic fungi, causing mycotic infections [6]. It has been reported that the human pathogenic species of Bipolaris are capable of infecting almost every site of the body and are emerging as potential pathogens of humans [7, 8, 9]. They cause both superficial and systemic infections with common clinical presentations, such as lung and skin infections, allergic sinusitis, onychomycosis, keratitis, and infections of the central nervous system [10, 11, 12].

Case Report

We report a case of 19-year-old female with complaints of discomfort on both feet. On examination, it was found that the skin of the toes of both feet was involved and affected. The infection evolved 8–9 years back. Clinical symptoms at the infection site include redness, dryness, excessive scaling, crusting, alopecia, and itching (Fig. 1a, b). The patient was a student by occupation, immunocompetent, and had no history of any underlying diseases, such as, AIDS, diabetes, and cancer. Other information collected from the patient include (a) habit of working in fields for prolonged hours, so the feet usually remain in contact with soil, (b) infection increases noticeably during the monsoon season which can be probably due to the more favorable conditions with high humidity in this season conferring it more conducive for the growth and colonization of B. cynodontis, and (c) no prior treatment.

Fig. 1.

A case of superficial fungal infection with the following symptoms: redness, dryness, excessive scaling, crusting, and alopecia (a, b); a moderately growing gray colony with aerial mycelium on modified SDA medium after incubation at 25°C for 1 week (c); reverse colony (d).

Materials and Methods

Ethical Approval

Ethical clearance for carrying out the present study was granted by Institutional Ethical Committee, University of Jammu, Jammu (No. RA/19/3118 dated August 23, 2019), which follows the guidelines issued by the Medical Council of India, India. Also, the consent of patient for sampling along with other details was taken in a detailed datasheet.

Sample Collection and Processing

After taking the consent and history of the patient, skin scraping was done carefully from the active margin of the lesion followed by mycological examination (cultural, microscopic, and molecular characterization) to identify the causal agent. The affected/suspected parts of the skin were cleaned thoroughly with 70% alcohol before collecting the samples. Skin scraping was done from the active margin of the lesion with sterilized blunt blade without injuring its surface. The samples were cultured in triplicates on modified Sabouraud dextrose agar (SDA) medium supplemented with chloramphenicol (0.05 mg/L). The isolations from the infected skin samples were repeated at least 3 times after an interval of approximately 15–20 days. The cultured Petri plates were incubated at 28 ± 2°C for 8–14 days.

For microscopic observations, the fungal cultures were mounted on slides using the lactophenol/lactophenol cotton blue stain. Using an ocular micrometer, the dimensions were determined for hyphae, conidiophores, and conidia. Microphotography of the lactophenol-mounted fungal cultures was done using a trinocular research microscope (Nikon model, Eclipse E400) fitted with a Samsung SDC-312 digital camera.

Identification

The recovered fungal species were identified by cultural observations (growth rate and culture characteristics, such as colony size, colony color, texture, exudates, and pigmentation on the surface or on reverse), and by microscopic and morphological observations. The identity of the causal agent was further confirmed by molecular characterization (PCR amplification and sequencing of ITS region), which was done at the National Centre for Microbial Resources, earlier as National Centre for Cell Science, Pashan, Pune (India).

DNA Extraction, PCR, and 16 s rRNA Sequence Analysis

The standard phenol/chloroform extraction method (Sambrook et al. [13]) was used for the isolation of the genomic DNA which was followed by PCR amplification of the ITS regions in which universal primers ITS1 [5′-TCC GTA GGT GAA CCT GCG G-3′] and ITS4 [5′-TCC TCC GCT TAT TGA TAT GC-3′] were used. For the purification of the amplified ITS PCR products, PEG-NaCl precipitation was used followed by direct sequencing on an ABI® 3730XL automated DNA sequencer (Applied Biosystems, Inc., Foster City, CA, USA) as per the manufacturer's instructions. In order to read each position at least twice, both ends were used for sequencing. As per Boratyn et al. [14], for assembly, Lasergene package followed by NCBI BLAST against sequences from type material was used for tentative identification.

Colonies on modified SDA medium after 10 days of incubation at 28 ± 2°C were woolly, white when young, and on maturity become grayish black, reverse black (Fig. 1c, d). Hyphae were septate and pigmented. Conidiophores were simple or branched measuring 95–345.8 × 3.8–4.1 µm, septate, flexuous, brown, pigmented, and geniculate at the upper part due to sympodial development (Fig. 2a, b). Conidia measured 19–52 × 7.6–9.5 µm, smooth, thin-walled, straight, or slightly curved, broader in the middle with rounded ends, hyaline when immature, pigmented (light to olivaceous brown) when mature with transverse distosepta, and without a protuberant hilum. The distosepta varied from 1 to 7. Hilum was inconspicuous (Fig. 2c, d) and a characteristic bipolar conidial germination, that is, germination from both end cells was observed (Fig. 2e, f); the end cell frequently swelled to form a thin globose vesicle which later gives rise to germ tube (Fig. 2c, d). The fungal isolate was identified as Bipolaris cynodontis (Marignoni), Shoemaker.

Fig. 2.

Microphotographs of conidiophore of B. cynodontis bearing conidia (a, b); conidia showing bulbous vesicle at the ends which later form germ tube (c, d); conidia showing bipolar germination, that is, germ tube development from both the ends (e, f). Scale bar, 10 µm.

Blast analysis of the ITS sequence (700 b) showed that it has the closest similarity to B. cynodontis (query cover: 100%, E value: 0.0, and percentage of similarity: 99.42%). Results were further confirmed using CBS and UNITE databases. The phylogenetic tree was also constructed using the maximum likelihood method, and the current isolate showed close relation (99% similarity) with B. cynodontis_CBS_109894 (Fig. 3). The sequence was submitted to NCBI GenBank (Accession No. MT994254).

Fig. 3.

Phylogenetic analysis of B. cynodontis SKI1 using the maximum likelihood method.

Discussion

Bipolaris is a dematiaceous and filamentous fungus belonging to the family Pleosporaceae of the class Pleosporales and has been reported to cause cutaneous and subcutaneous infections in humans. Earlier, a non-healing cutaneous ulcer due to Bipolaris species in a pancytopenia patient suffering from acute leukemia was reported by Straka and his coworkers (1989) [15]. Later, a report was established by Robb and coworkers in 2003 on superficial cutaneous infection caused by Bipolaris in 3 patients without predisposing medical conditions [16]. Similarly, Shafii et al. [17] also reported that Bipolaris species are capable of causing cutaneous and subcutaneous infections.

To the best of our knowledge, studies on the association of the current isolate, that is, B. cynodontis from clinical samples of humans are still meager and fragmentary. However, in a study carried out in the USA, out of 105 isolates studied, B. cynodontis was isolated from 9 clinical samples with 8.6% prevalence, thereby constituting a new global record [4]. Nath et al. [18] reported the first clinical case of B. cynodontis causing subcutaneous mycosis (chromoblastomycosis) on the right foot of a 58 years old non-diabetic male farmer from Assam (India). However, in contrast, during the present investigation, the infection was observed to be superficial with no effects observed in the tissues underlying the skin, thereby reflecting the potential of this rare opportunistic pathogen in causing skin infections of diverse types. From India, till date, there is no such report on the association of B. cynodontis and superficial mycoses of human skin samples. Therefore, from north India (J&K) and India, we are reporting the first case of superficial human skin infection caused by B. cynodontis.

As in the present case, the patient was a student by occupation and her feet usually remained covered by shoes during the school time that might be a probable reason for the initiation and propagation of fungal infection. Moreover, other predisposing factors, like working in fields for prolonged hours, rise in infection especially during the monsoon season and lack of treatment might have probably led to the congenial environment received by the fungal pathogen for further proliferation.

As non-dermatophytes are known to produce clinically similar lesions caused by the dermatophytes, it is difficult to identify the etiological agent only by morphological criteria [19]. Non-dermatophytic fungi, including Bipolaris species, are often considered as insignificant contaminant rather than etiological agents, infecting skin parts which are previously damaged by trauma or disease. However, they may actually act as a primary pathogen, as observed during present investigation.

Non-dermatophytic fungi, most of which live as saprophytes, it becomes difficult to distinguish the etiological agents from the contaminants especially when these fungal species are isolated from skin specimens. Therefore, it became necessary to check if similar colonies are obtained from clinical specimens and also whether the same etiological agent is identified from repeated cultures. In our case, the same fungal species was isolated from the repetitive culture of skin specimen confirming the identity of the identified causal agent. As this isolate, that is, B. cynodontis was isolated from the affected skin specimen, one should regard it as a potential causal agent requiring further mycological studies including a high degree of suspicion and careful examination of the clinical specimen instead of considering it merely a contaminant or clinically insignificant.

Conclusion

The present study supports that non-dermatophytic fungal species, including B. cynodontis, have the potential to cause superficial fungal infections and therefore should not be ignored. Early diagnosis, clinical awareness, and confirmation of etiological agents, such as Bipolaris infection by cultural, microscopic and molecular characterization followed by appropriate antifungal treatment at medical level and awareness for personal hygiene at the individual level can not only help in management of such infections effectively but can also halt the proliferation and spread of such infections.

Statement of Ethics

The patient gave written consent to publish the case including publication of images.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

University Research Scholarship (URS), University of Jammu, Jammu.

Author Contributions

Bharti Sharma contributed to case data acquisition, concepts and to manuscript writing. Dr. Skarma Nonzom contributed to concepts edited, reviewed and finalized the manuscript.