Original article / research
| Year :
2023 |
Month :
April
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Volume :
12 |
Issue :
2 |
Page :
PO31 - PO34 |
Full Version
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Evaluation of Fine Needle Aspiration Cytology versus Culture for Laboratory Diagnosis of Subcutaneous Mycosis
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Kalyani Prava Gouda, Pritilata Panda, Indrani Mohanty, Upasana Das
1. Associate Professor, Department of Pathology, PRM Medical College, Baripada, Odisha, India.
2. Professor, Department of Microbiology, PRM Medical College, Baripada, Odisha, India.
3. Associate Professor, Department of Microbiology, PRM Medical College, Baripada, Odisha, India.
4. Assistant Professor, Department of Pathology, PRM Medical College, Baripada, Odisha, India.
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Correspondence
Address :
Kalyani Prava Gouda, Pritilata Panda, Indrani Mohanty, Upasana Das,
Upasana Das,
Metro Manorama Complex, Kathagola Street, Cuttack, Odisha, India.
E-mail: drupasana80@gmail.com
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| | | ABSTRACT |  | : Introduction: Subcutaneous swellings are commonly seen in clinical practice having varied pathology from epidermal inclusion cyst to malignancy. Subcutaneous mycosis is a fungal infection of subcutaneous tissue and dermis which is more prevalent in workers of rural, tropical, and subtropical regions. The subcutaneous infection mostly occurs due to minor trauma which goes unnoticed. Fine Needle Aspiration Cytology (FNAC) followed by microbiological analysis is a cost-effective and simple procedure and can be considered as one of the main modalities for early diagnosis. Moreover, it gives an early presumptive identification of fungus, which helps the clinician in a timely therapeutic approach.
Aim: To study the efficacy of FNAC in the diagnosis of subcutaneous mycosis in comparison with culture method.
Materials and Methods: A cross-sectional study, including clinically diagnosed cases of subcutaneous mycosis was evaluated between March 2019 to February 2021 in a tertiary care hospital of Northern Odisha. FNAC samples from all the cases of subcutaneous mycosis were studied cytologically and microbiologically. Cases were analysed and further evaluated on the basis of their age, sex, detailed clinical history, and presentation. FNAC showed the presence of fungal elements in 20 cases. Microbiological evaluation of these samples were carried out.
Results: Out of the 50 cases, 20 (40%) were diagnosed to be subcutaneous mycoses and the majority were males 16 (80%). The most common site of involvement was the lower limb (60%) followed by upper limb (30%). Almost all the cases were from rural areas with a history of trauma present in 11 cases (55%). Out of 50 clinically diagnosed cases, cytological examination revealed fungal element in 20 (40%) cases. The most common cytological diagnosis was abscess with neutrophilic infiltration seen in 13 cases (65%) followed by granuloma in 7 cases (35%). All the 20 samples were further evaluated in conventional methods like Potassium Hydroxide (KOH) mount and culture. Fungal agents were isolated in 13 (65%) cases. Cladosporium spp. was the most common isolate in 5 cases (25%).
Conclusion: Subcutaneous mycosis is a rare disease. FNAC can be used as a rapid and cost-effective screening tool in the diagnosis of such cases as it is more sensitive. Though culture is the gold standard test, it is less specific. |
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| Keywords
: Abscess, Dematiaceous fungi, Fungal infection, Granuloma |
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| DOI and Others
: DOI: 10.7860/NJLM/2023/57361.2720
Date of Submission: Apr 26, 2022
Date of Peer Review: Jun 10, 2022
Date of Acceptance: Oct 19, 2022
Date of Publishing: Apr 01, 2023
AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? No
• For any images presented appropriate consent has been obtained from the subjects. Yes
PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Apr 27, 2022
• Manual Googling: Jul 29, 2022
• iThenticate Software: Oct 15, 2022 (5%)
ETYMOLOGY: Author Origin |
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INTRODUCTION |
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Subcutaneous mycoses are infections caused by saprophytic fungi, which get inoculated to the site of infection usually by penetrating injury. Such infections are more common in rural and agricultural populations (1). Subcutaneous mycoses may be of various types like eumycetoma, chromoblastomycosis, phaeohyphomycosis, etc., (2). As it usually presents with localised swelling without any obvious signs of inflammation, more often clinically it is mistaken for other benign lesions like an epidermoid cyst, sebaceous cyst, or cold abscess (3). FNAC followed by histopathological examination is a principle diagnostic method required for the diagnosis of subcutaneous mycoses. It is reliable and less time-consuming than culture (4). Though modern analysis including Polymerase Chain Reaction (PCR) is now available, it is time-consuming, costly and needs a special laboratory setup. So, for early presumptive diagnosis, cytological evaluation of granulomatous lesions with or without giant cell reaction, necrotising inflammation with a careful search for fungal elements, histopathological examination, subsequent special stain, and culture to demonstrate and identify the fungus wherever possible provides indisputable evidence of invasive infections.
The biopsy is painful and can cause scarring and requires more time and may be done in cases where the representative sample is not sufficient or correlation is required. Because of its morphological diversities, many of the fungi are seen under light microscopy by conventional diff-quick and Haematoxylin and Eosin (H&E) stain. Special stains like gram stains, Periodic Acid Schiff (PAS), and Giemsa stains may be used for the identification of fungi in cytology specimens. Fungal elements are usually present in hyphae, budding yeast, endosporulating spherules, or in combinations along with tissue reactions depending on their virulence (4).
Keeping in view the above background, the aim was to study demography, clinical presentation of subcutaneous mycoses, their diagnosis by FNAC and confirmation by culture isolation. As FNAC is a common and simple outpatient department procedure, it was used to diagnose subcutaneous mycosis. Biopsy was done in equivocal cases. Culture was used in the diagnosis and identification of the causative agent causing subcutaneous mycosis. The objective was to study the efficacy of FNAC in the diagnosis of subcutaneous mycosis.
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Material and Methods |
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A cross-sectional study was conducted on clinically diagnosed cases of subcutaneous mycosis presenting as subcutaneous swellings in the Department of Pathology and Microbiology in a tertiary care hospital of PRM Medical College, Baripada, Odisha, India from March 2019 to February 2021 with a total number of 50 cases. The ethical practice of conducting research with IEC number 1 1/Dt. 19.06.2019 has been implemented.
Inclusion criteria: All cases of subcutaneous swellings clinically diagnosed as subcutaneous mycosis, reported in the section on cytopathology during the study period of March 2019-February 2021.
Exclusion criteria: Follow-up cases of subcutaneous swelling due to non infectious aetiology, malignancy, deep fungal infection were excluded from the study.
Procedure
Patient clinical data was collected from records maintained in cytology section and analysed with respect to age, sex, site of involvement, duration of the lesion, predisposing factors like thorn injury, and other co-morbid conditions like diabetes mellitus, chronic non healing ulcers, high blood pressure and malignancy.
The subcutaneous swellings were examined. Most of them presented as a cystic mass and nodule. Patient was explained about the procedure of fine needle aspiration. A 23 G needle was inserted in the subcutaneous swelling and with a 10 mL syringe, aspiration was done. The aspiration material was usually fluid or pus and spread over a glass slide. In case of fluid aspiration of 2 mL, sample was centrifuged and from the deposits, smears were prepared. Two cytology smears were prepared in each case. One was kept for air drying and stained with Leishman and Giemsa stain. Another slide was kept for fixation by methanol and stained with H&E stain.
In the Department of Microbiology, all the samples were examined under direct microscope in KOH mount and gram staining. They were inoculated into blood agar, MacConkey agar, and two tubes of Sabouraud Dextrose Agar (SDA) with antibiotics incubated at 37°C and 25°C, respectively. The growth in blood agar and MacConkey agar were examined after 24-48 hours and identified by conventional methods. Growth in SDA was examined every alternate day for the first week and thereafter twice a week for 6 weeks. The fungal growth was identified based on the time taken for growth to appear, colony morphology, and microscopic appearance.
The cytological features were studied. The equivocal cases sent for biopsy were traced from records and histopathological features were noted down. Special stains like PAS stain was done from the blocks of subcutaneous mycosis demonstrating fungus.
Statistical Analysis
Data entry was done using an MS Excel sheet. Categorical variables were expressed using frequency and percentages. Quantitative variables were expressed using the mean. Demographic data like age, sex, etc., were expressed in percentages. Hypotheses are formulated as follows.
H0: There is no significant difference between the effectiveness of both the tests.
H1: There is significant difference between the effectiveness of both the tests. The t-test was applied for hypothesis testing.
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Results |
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Out of the 50 cases, 20 (40%) were diagnosed to be subcutaneous mycosis (Table/Fig 1) and the majority were males 16 (80%). The most common affected age group was between 20-60 years with a total number of 15 (75%) cases from this age group (Table/Fig 2). The most common site of involvement was the lower limb in 12 (60%) cases followed by the upper limb in 6 (30%) cases and history of trauma in 11 (55%) of total cases (Table/Fig 3). Almost all cases were from rural areas and were agricultural workers. There were no other co-morbidities like diabetes mellitus, malignancies or Human Immunodeficiency Virus (HIV) etc. Two cases had additional atypical presentations; one case presented with multiple erythema like lesions over the head and neck and upper arm and another case presented with multiple plaque-like lesions over the tongue. Abscess with neutrophilic infiltration and hyphae were the most common cytological features in 13 (65%) cases, followed by granuloma with giant cell reaction and hyphae in 7 (35%) cases in the 32subcutaneous mycosis cases (Table/Fig 3). A comparative analysis was done between different types of tests (Table/Fig 4). (Table/Fig 5) demonstrated a patient who presented with a subcutaneous swelling over the ulnar border of right hand. The fungal structure was identified in H&E, Diff-Quick stain, and Gram stain. The most common fungal structure identified in this study was branching septate hyphae and most of them were dematiaceous [Table/Fig-6,7]. Histopathological examination was done in 6 equivocal cases (30%) out of 20 subcutaneous mycosis cases. Cases revealed non specific lymphohistiocytic infiltration, few giant cells and presence of branched, thin, septate hyphae intermingled with each other. PAS stain faintly demonstrated the fungal hyphae (Table/Fig 8).
Out of 20 cases of cytologically diagnosed subcutaneous mycoses, fungal isolation was successful in 13 (65%) cases (Table/Fig 4). Cladosporium species [Table/Fig-9,10] was the most common followed by Fonscasea spp. and Curvalaria spp. Bacterial isolates like Staphylococcus aureus without any fungal element were found in 4 (20%) cases. There was no growth in 3 (15%) cases. May be fungal element had lost its viability either due to delayed transport to microbiology laboratory or the patient might have received treatment prior to collection of sample and fungal element could not be cultured. Staphylococcal infection was the superadded infection observed in 4 (20%) cases.
(Table/Fig 11) represents the result of the t-test applied with p-value of 0.0047. Therefore, the null hypothesis is rejected and the alternate hypothesis is accepted that there is a strong significant difference between test methods (cytology and culture).
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Discussion |
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Pathogenic fungi or mycosis may infect various tissues ranging from skin to subcutaneous tissue and may disseminate to deeper organs like brain, heart, lungs and liver. The term subcutaneous mycosis describe the lesion limited to dermis and subcutaneous tissue and elicit a chronic, localised inflammatory response at the inoculation site of the aetiological agent.
Subcutaneous mycoses cases are mostly seen in the hot and humid climate of the tropical region. In Odisha, the geographically northern part is a hilly tropical region with more forest land where the subcutaneous fungal infection is a common occurrence. Authors have studied 50 cases within 2 years. Verma S et al., also reported 70 cases over 4 years in the Himalayan region (3). Maybe the number of cases is under-reported because of the callousness of the people due to which they do not take such lesions seriously and many going for indigenous methods of treatment. The primary fungal infection occurs due to traumatic penetration. Usually, this traumatic injury occurs in the barefooted population of the world, agricultural workers, mountain hikers, and gardeners (4). Nearly all other studies showed involvement mostly of lower limbs. Most of our patients were forest dwellers or living in villages adjoining forests whereas Yahya S et al., demonstrated agricultural occupation as a risk factor (5).
Our study demography revealed most patients were in middle age group (75%). Micro-trauma leads to implantation of fungi in the subcutaneous tissue leading to chronic inflammatory tissue reaction. History of minor trauma was found in 55% cases in hard-working daily labourers whereas others could not recall.
Clinically, most of the patients of subcutaneous mycoses present with cystic swelling, plaque like lesion, ulcers and warty verrucous growth (5). In present study, all 20 cases of subcutaneous mycoses cases presented as cystic or solid subcutaneous swellings. Cystic swelling was the most common presentation in other studies too [6-9]. Carrion in 1950, described five different clinical forms of Chromoblastomycosis presenting as nodule, tumorous, verrucous, cicatricial and plaque type.
Most of the fungi produce hyphae- septate or aseptate, arranged as branched intertwined hyphae. Dimorphic fungi exist both in yeast and hyphae form depending upon the environmental condition. In the present study, the distinguishing parameters to differentiate between different fungi were based on budding, presence of capsule, septa and branching pattern. Cytological evaluation of all the aspirated samples showed the presence of fungal elements in 20 cases (40%). There are very few studies on FNAC of the samples of subcutaneous mycosis for correlation [10,11]. Most of the studies were carried out on biopsy and culture.
Fungal hyphae can induce either granulomatous inflammation or suppurative abscess (12),(13). On aspirated material, H&E and Giemsa staining demonstrated the presence of fungal hyphae as branched, parallel hyphae with or without septation and most were dematiaceous. The background contained polymorphs and histiocytes in suppurative lesions and granuloma lesions featured multinucleated giant cells, eosinophils and lymphocytes. Verma S et al., and Priyadharshini G et al., demonstrated giant cell reaction with granuloma as their most common finding on histopathological examination (3),(10). Subhashini R and Bhat RV observed heavy eosinophillic infiltration in 36% of cases (4).
This study revealed Cladosporium spp. (25%) as the most common fungal isolate followed by Fonsecaea spp. and Curvularia spp. (15% each) whereas others isolated Phialophora spp. and Fonsecaea spp. as the predominant isolate (Table/Fig 12). Verma S et al., found chromoblastomycosis as the most common organism causing subcutaneous mycosis (3) Yahya S et al., observed subcutaneous mucormycosis as the most common case followed by chromoblastomycosis. Culture revealed Basidiobolus ranarum in mucormycosis and Phialophora in chromoblastomycosis as the commonest aetiological agent (5). All the fungal isolates were dematiaceous fungi; hence 65% of the subcutaneous mycoses cases were diagnosed as phaeohyphomycosis in the present study. Subhashini R and Bhat RV also identified phaeohyphomycoses as the most common type of subcutaneous mycosis. Priyadharshini G et al., found phaehyphomycosis as the only causative organism in their study [4,10].
Elephantiasis and lymphoedema are some rare complications associated with chromoblastomycosis due to chronic inflammation followed by fibrous scarring. Verma S et al., also reported squamous cell carcinoma as a long standing complication of chromoblastomycosis (3).
While analysing the efficacy of FNAC in the diagnosis of subcutaneous mycosis, 20 cases showed presence of fungal element whereas culture method isolated fungus in 13 cases. There is significant statistical difference between the tests. FNAC was found to be sensitive and can be used as a screening procedure. Culture, the conventional method of diagnosis is specific, but less sensitive as viable fungus presence is essential for the growth. All the cases of subcutaneous mycosis were followed-up and they had shown improvement after taking antifungal drugs like Itraconazole for 3 months.
Limitation(s)
The limitation of the present study was that the histopathological study could not be done in all the cases as it was an invasive procedure.
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Conclusion |
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As Fine Needle Aspiration Cytology (FNAC) is more sensitive, it is a useful tool for early diagnosis of subcutaneous mycosis as it is cheap and less time-consuming in experienced hands. Subcutaneous mycosis is usually more prevalent in hilly forest areas with a tropical climate and as this region has the same topography; more screening is required along with awareness among the natives. The present study was done to assess diagnostic efficacy of FNAC in the detection of subcutaneous mycosis in a new medical college in Northern Odisha with limited resources.
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| TABLES AND FIGURES | |
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