An overview of mycetoma and its diagnostic dilemma: Time to move on to advanced techniques

Gross examination, microscopy and culture

The specimen is collected by pressing the sinus from suspected lesions to collect grains which are then subjected to gross examination, microscopy and culture. The gross examination includes an assessment of the colour, size, shape and texture of grains. For example, black grains are suggestive of eumycetoma, and red grains are seen in Actinomadura pelletieri. Grains of Nocardia asteroides and Nocardia brasiliensis are smaller in size while large-sized grains are observed in Madurella spp. and Actinomadura madurae. Grains harbouring Streptomyces somaliensis and Madurella mycetomatis can be hard in consistency though grains of other pathogens are mostly soft in consistency.^18,19

The various techniques of microscopic examination include Gram stain, modified Ziehl Neelsen (Z-N), or haematoxylin and eosin (H and E) stain for actinomycetoma and potassium hydroxide (KOH) mount, histopathological stains like H and E, Gomori’s methenamine silver (GMS) or periodic acid Schiff (PAS) stains for eumycetoma. In 2009, Alam et al. reported five cases of mycetoma from India presenting with ulcers/purulent discharge admixed with black granules. Haematoxylin and eosin stained sections and Gram stain showed thin filamentous bacteria (actinomycetoma) and thick club-shaped structures (eumycetoma).¹⁸

Culture is required to identify the organism properly, as morphological features of grains can be similar in many of the agents causing mycetoma. The various culture media required are Sabouraud Dextrose Agar with and without antibiotics, 5% sheep blood agar, brain heart infusion agar, Lowenstein Jensen media in multiple sets. Each set of culture media is incubated at both 25°C and 37°C and observed for growth which occurs usually after 7-10 days,²⁰ though delayed growth may be observed especially in case of fungal pathogens, hence culture media needs to be incubated for 4-6 weeks before reporting it as negative.¹⁹ Padhi et al. detected 13 cases of mycetoma by histopathological examination but in only 8 /13 cases growth was seen on culture (though in 1/5 of remaining cases, culture was not done).⁵ A flowchart of routine diagnostic workup is demonstrated in Figure 1 which can be done in resource-poor settings with no access to molecular facilities.²

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Figure 1:

Diagnostic algorithm for mycetoma²

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Cytodiagnosis

Cell block technique can provide us with the combined advantages of routine histopathology and fine needle aspiration (FNA) direct smears. Aspirated material from suspected lesions can be used to prepare a fixed cell block. This technique is described as safe, rapid, and effective by Yousif et al. in a study done on 240 patients.²¹ The authors reported sensitivities of 85.7% and 87.5% for actinomycetoma and eumycetoma, respectively utilising this technique and there was no difference statistically in the findings obtained by the histopathological sections and cell block. The cell blocks have the added advantage of being stored for the prolonged duration for future studies and hence provide a ray of hope for researchers working on mycetoma.

Mycetoma Research Centre of the University of Khartoum, Sudan conducted a study using FNA from lesions for the identification of grains and tissue reaction. Surgical biopsies and histopathological examinations were performed if FNA was found to be negative in a suspicious case. In the study, 3177 (47%) patients underwent FNA for cytological examination and the findings were compatible with Madurella mycetomatis in 2379 (75%) cases, Actinomadura madurae in 316 (10%) cases, Streptomyces somaliensis in 277 cases (7%), Actinomadura pelletieri was uncommon and was detected in 39 (1%) cases.²²

Radiology

Extension of the lesions in bone and other tissues can be determined by magnetic resonance imaging (MRI), X-rays, and tomography.²³ In MRI, ‘dot in circle’ is a radiologically relevant sign of mycetoma.²⁴ This sign is described as hypointense foci within the hyperintense spherical lesions that comprise granulomatous tissue surrounding fungal elements/bacterial colonies. Limited availability of MRI and lack of expertise are hindrances in diagnosis, especially in rural areas where the disease is prevalent. In the context of ultrasonographic findings, thick-walled cavities with hyper-reflexive echoes representing grains may be seen in eumycetoma while in actinomycetoma fine, closely aggregated hyperechoic foci are seen at the bottom of rounded lesions.¹⁹

Serology

Serological tests are of limited value in the diagnosis of mycetoma as most of the tests utilise cell extracts instead of exoantigens and cross-reaction can occur with other diseases. The various serological methods that have been used are the complement fixation test, immunodiffusion, counter-immunoelectrophoresis, western blot and enzyme-linked immunosorbent assays.²

Molecular diagnosis

Though molecular techniques are heading towards advancement in routine diagnosis of mycetoma, these are costly methods and still lack standardisation in many laboratories. These methods can also be used for epidemiological studies and generating accurate therapeutic options thus minimising the use of combination drugs. In 2017, Bitan et al. reported four new cases from Israel and also documented 17 other case reports between 1942 and 2015 from the same place. They included pan-fungal polymerase chain reaction (PCR) according to the protocol described in http://sites.biology.duke.edu/fungi/mycolab/primers.htm and pan bacterial PCR (16S rRNA gene PCR and sequencing) in diagnosis apart from conventional methods.²⁵ Out of four, two culture-negative cases were successfully detected by PCR.

Rolling circle amplification (RCA) that uses species-specific padlock probes, loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) proved to be highly useful techniques for the diagnosis of mycetoma in studies done by Ahmed et al. ^26,27 The basic methodology of RCA involves the detection of specific sequences of nucleic acid and enzymatic amplification of circularised oligonucleotide probes under isothermal conditions.²⁸ It is a highly specific, simple, rapid, single-day protocol and is of low cost. Sixty-two isolates were identified by Ahmed et al. utilising the RCA technique and the results were 100% specific with zero cross reactivity.²⁷ The major limitations of RCA are the culture of the pathogen and the requirement of PCR amplification.^26,27

LAMP technique relies on the utilisation of four primers that identify six regions in the target DNA, followed by amplification via auto-cycling and strand displacement with a specialised DNA polymerase.^29-31 RPA is another rapid detection isothermal technique which can be utilised in places where mycetoma is endemic. The risk of contamination was reported to be lower than LAMP by Ahmed et al. Both RPA and LAMP techniques (sensitivity and specificity=100%) were found to be equal in performance to PCR in detecting Madurella mycetomatis directly from clinical samples.²⁶

MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry)

MALDI-TOF MS is a high throughput proteomic technique that requires co-crystallisation of an analyte with a matrix that absorbs laser striking it and converts the analyte molecules into the gas phase. The ions thus formed, travel, and reach the detector at a speed inversely proportional to their mass/charge (m/z) ratio. A signal is created indicating m/z ratio (x -axis) and ion intensity (y -axis), according to the time taken to hit the detector by each of the ion particles.³² By incorporating this method, rapid and accurate identification of etiological agents of eumycetoma is possible thus eliminating the need to wait for culture reports. Fraser et al. tested 57 organisms corresponding to 10 different species from confirmed cases of subcutaneous pedal masses and eumycetoma, identified previously by PCR amplification and sequencing of Internal Transcribed Spacer 1 (ITS 1). They demonstrated accurate identification of all of the test organisms with the help of this technique, obtaining log scores more than 1.8, 1.9 and 2.0, in 100%, 90.4% and 67.3% of isolates respectively.³³ Recently, dermatophytic mycetoma of scalp was reported by Diongue et al. which is a rare condition. The causative organism was identified as Microsporum audouinii (atypical strain) by MALDI-TOF MS with a Biotyper score of 2.022. They further confirmed the organism by nucleotide sequence analysis of the ITS region of the rRNA gene.³⁴

MALDI-TOF MS is less labour-intensive, more cost-effective, and gives faster results in approximately 1 day, as compared to partial (500-bp) 16S rRNA gene sequencing. Buckwalter et al. were able to detect Nocardia species (90%) and other aerobic actinomycetes (51%) by MALDI-TOF with the help of a custom enhanced library, and some additional processing steps.³⁵

While utilising MALDI-TOF technique in the identification of genus Nocardia, spectra adequate for identification cannot be obtained using only processes for general bacteria because their cell wall contains aliphatic acid such as mycolic acid, hence Segawa et al. proposed another method, Nocardia extraction method in Department of Clinical Laboratory at Chiba University Hospital (NECLC). In this method, the ethanol-formic acid extraction technique is utilised, silica beads are added as a part of the high-temperature extraction method, and extraction of formic acid is done within 10 minutes.³⁶

The various advantages and disadvantages of different techniques used in the diagnosis of mycetoma are summarised in Table 2.^{2,7,13,18,21,26,27,35-38} Recently, an interesting survey was done by Hay et al. based on responses given by 23 clinicians and mycologists on the diagnostic techniques of fungal neglected tropical diseases. Percentage positive response of participants regarding the optimal diagnostic method in well-equipped settings versus diagnostic methods of use in peripheral clinics and laboratories for mycetoma (in terms of percentage) is depicted in Table 3.³⁹ In the Indian context, newer technologies are not available everywhere and much needs to be done in this field. Making clinicians aware and promoting research and development in mycetoma diagnostics is important in the current situation.

Key points of the review include limitations of the newer techniques

1. Microscopy is relevant and irreplaceable for a basic workup to differentiate Actinomycetoma from Eumycetoma.

2. Teamwork between clinicians, microbiologists and pathologists have to be strengthened as it plays a vital role in the diagnosis of mycetoma.

3. Molecular methods can make the diagnosis easy, timely and definite. Hence it will help the clinicians to avoid prescribing combination drugs, thus helping to fight emerging resistance.

4. Newer diagnostic methods like MALDI-TOF, can accurately diagnose various pathogens causing mycetoma but are still sidelined and not being incorporated at places with good access to laboratory facilities.

5. The newer techniques are mainly limited by the cost, especially in resource-poor regions where mycetoma is more common.