Prevalence of Aspergillus Infection in Various Clinical Samples with a Special Reference to Black Grain Eumycetoma Caused by Aspergillus nidulans in a Tertiary Care Hospital in Eastern India

1. This original research article represent  a prospective study  to determine the prevalence of Aspergillosis  in superficial, systemic & subcutaneous infections.
2. In this study diagnosis & speciation of Aspergillus isolates has been done to ascertain the  significance of specific fungal isolate in causing local and systemic pathogenesis.
3. This study has also revealed Aspergillus nidulans as a causative agent of Black Grain Eumycetoma in Indian sub=continent.

Among invasive aspergillosis, pulmonary aspergillosis & Rhinosinusitis are most common. Pulmonary aspergillosis mainly affects people with compromised immune status, underlying lung disease or asthma through spore inhalation. Lower WBC count in people having chemotherapy, organ transplant or leukaemia render them more susceptible to invasive Aspergillosis. In this context, BAL fluid is an ideal sample to be processed.

*TDO - Total Dystrophic Onychomycosis, PSO  - Proximal Subungual Onychomycosis , DLSO -  Distal Lateral Subungual Onychomycosis

Table (2).    Age-wise distribution of Aspergillosis (Total 32 isolates among 300 samples) (n=32)

Table (3). Prevalence of various Aspergillus isolates (N=32)

Table (4). Findings of Madura foot isolate (Aspergillus nidulans) 

DISCUSSION

Here incidence of hand nail involvement is more frequent than toenails. Typically, there is early involvement of lamina and painful peritonitis without pus. Unlike dermatophytes, non-dermatophyte moulds (Aspergillus spp.) are usually not keratolytic. Therefore, previous keratin destruction by dermatophyte, trauma or another nail disease can favour the invasion of the nail bed by Aspergillus spp. as a secondary invader [7].

Aspergillus spp. are insensitive to terbinafine or itraconazole, which are usually applied for dermatophytes. A Standard treatment for onychomysosis due to Aspergillus sp.  has not yet been established & perfect cure is rather difficult to obtain.

A review of 42 epidemiological studies due to Aspergillus sp. varies between <1% & 35% of all cases of onychomycosis in the general population. It is very uncommon among children & adolescents.

 Aspergillosis in the urinary tract is a rare disease. In most cases invasive aspergillosis in the urinary tract is due to nosocomial infection; diagnosed in immunocompromised patients such as organ transplant, hematologic malignancies & immune-suppressive therapies, following instrumentation.

Discussion on Aspergillus nidulans, the causative agent of Eumycetoma (Madura foot):

Clinical discussion:Mycetoma is a slowly progressive, chronic subcutaneous granulomatous infection of skin & subcutaneous tissue clinically presented tumefaction & oozing granules from multiple discharging sinuses. It develops after traumatic inoculation with soil contaminated by causative agents, mostly affecting the lower extremities &  exposed areas over the hands. The disease is indolent & disfiguring and may involve adjacent fascia & bony structures after a long duration. Complications include fractures of infected bone & bacterial superinfection.  Skin shows a wooden fibrotic Induration due to dermal sclerosis, draining mycetomas are often superinfected by Staphylococcus sp. & streptococcus sp. Patients are otherwise in good health with no satellite adenopathy.

Etiological Discussion: Unlike the usual fungal agents of black grain eumycetoma [8], here the isolate has been diagnosed as Aspergillus nidulans  ( also called Emericella nidulans). The isolate is a homothallic fungus i,e able to self-fertilize & form fruiting  bodies. Grains represent the aggregates of fungal   hyphae, 2-6 μm in diameter in eumycetoma.

Incidence & Epidemiological Discussion: mycetoma is endemic in tropical & subtropical countries between latitudes 15⁰N & 30⁰S; known as the Mycetoma belt. The highest incidence is found in Sudan, Venezuela, Mexico & India. The first attempt to map the distribution of mycetoma globally was made by Abbott (1956) by studying 1321 cases of mycetoma in Sudan⁹. Sudan is considered the most endemic country & 70% of cases of mycetoma (among > 7000 patients) have been identified due to Madurella mycetomatis in its capital, Khartoum [10].

Extreme climatic conditions in endemic areas contributes to survival of causative agents, more commonly affect young men aged 20-40 yrs. Male:Female ratio of mycetoma cases is 3.5 : 1.

In Southeast  Asia, India, and Pakistan the ratio of Eumycetoma & Actinomycetoma is 35% & 65% respectively. In India, most cases of mycetoma are found in Tamil Nadu (Madurai) and the moist southern part of Rajasthan. In India Mycetoma due to A. nidulans is rare, with only a few case reports of white-grain eumycetoma by this isolate [11,12].

Laboratory Discussion: Isolation of black grains from the tissue sample & 20% KOH mount of saline-washed crushed grains remains the crucial step. Actinomycetoma was excluded by modified ZN staining (negative). The study of granule colour, texture, size & presence of hyaline/pigmented hyphae helps determine causative agents (here it is hyaline hyphae). Isolation is slowly growing in culture, so meticulous daily observation of cultural media is needed.

LPCB staining from culture revealed the characteristic structures of Aspergillus nidulans. H 15u"> lle cells are thick-walled globose cells in shape, emerge from cleistothecium, and are specialized for versatile dispersion & re-genesis. These cells can grow into hyphae that develop into spore-producing colonies.  During sexual development these multinucleate cells can inherit nuclei from both parents, indicating that they may serve as genetic backups.                         

Instead of conventional slide culture by cornmeal agar block; banana peel culture method used for easy identification of late-sporulating human fungal pathogens [6].

Banana (Musa sapientum) peels are high quality & cheap source of carbohydrates, & minerals & it promotes growth of reproductive structures. LPCB staining from Banana peel culture  showed  the same entity like a slide culture, producing fruiting bodies, which is characteristic for Aspergillus nidulans.

Several studies on Aspergillosis have been conducted. In one study Felix Bongomin et al. [13] studied 42 cases of onychomycosis & prevalence of aspergillosis was 7.7% of the proportion of NDMO. A. flavus, A. terreus, & A. niger were the most common etiological sp.  Another epidemiological analysis of onychomycosis was conducted at Montpellier University Hospital, France (1991-2019) by Laetitia Laroche et al. [14] revealing nail aspergillosis prevalence of 16.9 % of cases. Analysis of clinical reports of 102 patients with ungual aspergillosis (men/women ratio: 0.67; mean age 56.3 years) identified cardiovascular, endocrine, cancer & skin diseases as contributing factors.

Though commonest species implicated in invasive aspergillosis is Aspergillus fumigatus, but in developing countries A. flavus has been isolated comparatively at higher frequency from sino-orbital Aspergillosis [15],  which is closelycorroborated with our study revealing Aspergillus flavus as most prevalent pathogen causing  invasive Aspergillosis.                                                                                   

CONCLUSION

It is nearly impossible to avoid exposure to Aspergillus sp. A. flavus & some other isolates can produce aflatoxin (mycotoxin), having carcinogenic potential. Environmental stress can upregulate aflatoxin production by the fungus, which can occur when the fungus is growing on damaged plants. Galactomannan (GM), a major component of the Aspergillus cell wall; is released during invasive disease. The level of circulating serum or plasma GM is indicative of fungal burden in the host. A positive GM result should be considered along with microbiologic culture, histology of biopsy specimen & radiographic evidence. The use of GM EIA & qPCR assay in conjunction with culture-based diagnostic methods applied to BAL fluid could facilitate accurate diagnosis & more timely initiation of specific therapy. Posaconazole, an extended-spectrum Triazole antifungal agent is effective against Aspergillus.

The global emergence of azole-resistant A. fumigatus isolates notoriously limits the therapeutic options. Although specific mutations in CYP51A are the main cause of azole resistance, there is a new wave of azole-resistant isolates with wild-type CYP51A genotype challenging the efficacy of the current diagnostic tools. Therefore , applications of whole genome sequencing are increasingly gaining popularity to overcome such challenges.

Diagnosis of invasive aspergillosis remains difficult in that clinical manifestations are not specific; radiologic findings can be suggestive but not pathognomonic. Along with definite LPCB  morphology from fungal growth, histologic demonstration of invasive hyphae represents proven invasive fungal diseases .

Ethical approval:

 The study was conducted after meeting all ethical considerations as per the declaration of Helsinki.

Conflict of Interest: There is no conflict of interest involved in this study.

Funding Sources:  None. Author funded.

Availability of data and materials

Acknowledgement :

The authors thank all other faculties of the Department of Microbiology of NRS Medical College &

hospital along with the Medical Laboratory Technologists in the Mycology laboratory who helped a lot during the conduction of this study. The authors also thank the patients and their next to keen, who consented and willingly participated in this study.