Human actinomycetoma caused by Actinomadura mexicana in Sudan: the ﬁrst report

Mycetoma is a localized, chronic, granulomatous disease that can be caused by fungi (eumycetoma) or bacteria (actinomycetoma). Of the 70 different causative agents implicated in mycetoma worldwide, Actinomadura madurae is the only one that causes multiple cases on all continents. Recently, new Actinomadura species were described as causative agents of human mycetoma. One of these new causative agents was Actinomadura mexicana , which was identiﬁed in Latin America. Here we demonstrate that this causative agent is not conﬁned to Latin America and that it is also a causative agent of actinomycetoma in Sudan. The disease was managed by antibiotic treatment alone and resulted in complete cure after 6 months of treatment, which is quick when compared with actinomycetoma cases caused by other Actinomadura species.


Introduction
2][3][4] The disease develops after the traumatic inoculation of the causative agent into the subcutaneous tissue. 5The feet are most commonly infected, 6 however, other parts of the body are not exempt. 7The disease is characterized by painless, subcutaneous swellings and draining sinuses. 8The population at risk is mainly adult and 20-40 y of age. 7Treatment is dependent on the causative agent and consists of antifungal treatment combined with surgery for eumycetoma or antibacterial treatment for actinomycetoma.The duration of the medical treatment is long and takes at least 6 months and often up to 2 y.The duration of the treatment is dependent on the extent of the lesion, but is probably also linked to the aetiology of the causative agent.Therefore accurate species identification is important in the proper treatment and management of mycetoma patients.In this communication, we report for the first time in Sudan a case of actinomycetoma caused by Actinomadura mexicana; the patient was cured after short-term antibiotic treatment.

Case report
The patient is a 26-year-old male, a teacher from Al Gezira State, Central Sudan who presented to the Mycetoma Research Centre (MRC), University of Khartoum, Sudan in December 2018 with a foot swelling.His condition started 6 months prior to presentation, with a small swelling on the dorsal aspect of the left foot first web space.It was of gradual onset and progress.He denied any history of local trauma at the lesion site and he had no local pain.He has no family history of a similar condition.His past medical, social or geographic history was non-contributory to his present condition.He was not on long-term medication.
On clinical examination, the patient looked well, was not pale and neither jaundiced nor cyanosed.He had a normal pulse rate of 76/min, respiratory rate of 17/min, blood pressure of 110/75 mmHg and temperature of 37°C.A systematic clinical examination was normal.Local examination revealed a localised lesion, 3 cm in diameter, on the dorsal aspect of the left foot first toe web.The swelling was not tender, with normal temperature, firm in consistency, localised and attached to the skin and deep structures.The skin was normal and no local sweating or sinuses were noted.The regional lymph nodes were not enlarged.

© The Author(s) 2020. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.
All rights reserved.For permissions, please e-mail: journals.permissions@oup.comTransactions of the Royal Society of Tropical Medicine and Hygiene His renal and hepatic function tests, as well as his full blood count, were within normal limits.X-rays of the left foot were reported as normal (Figure 1), but the ultrasound examination showed multiple cavities with thick walls that contained fluid, with sharp echogenic grains and intact bone, in line with the diagnosis of eumycetoma (Figure 2).
For confirmation and identification of the causative organism, a deep surgical excisional biopsy was performed.The grains present in the biopsy were white to yellow in colour and small in size.They resembled those of Actinomadura madurae and to lesser extent Streptomyces somaliensis.The grains were smooth in consistency and easily crushed.
The biopsy was processed for histopathological examination and the sections were stained with haematoxylin and eosin (H&E).Microscopic examination showed multiple dark blue to purple-stained grains surrounded by granulation tissue (Figure 3A).The grains looked opaque at the periphery, with deep blue staining.The centres of the grains were less densely stained.The peripheries of the grains were eosinophilic.There were marked histiocytic and mixed inflammatory cellular infiltrates, in line with A. madurae (Figure 3B).
After separation from the surgical biopsy, the grains were washed three times in normal saline and then cultured in yeast extract media and Lowenstein media at 37°C.After 10 d, red to pink colonies were visible with a convex surface.The colonies were very tough in consistency and wrinkled in shape (Figure 4A).These colonies resembled those of A. madurae (Figure 4B) and were identified as such.
To confirm the identification of the causative agent, DNA from the isolate was extracted and the 16S ribosomal RNA region was amplified and sequenced using forward primer (5 -CTTTACGCCCARTRAWTCCG-3 ) and reverse primer (5 -AGAGTTTGATCMTGGYTCAG-3 ).After analysing and blasting the sequence, it was found to be 100% identical to A. mexicana strain A290 16S sequence NR_025110.1.The obtained MRC/A-04 16S sequence was deposited in GenBank with the accession number MT302842.
On January 2019, the patient started on an antimicrobial combination of 2 g co-amoxiclav (875 mg amoxicillin and 125 mg clavulanic acid) and two tablets of 1840 mg co-trimoxazole daily.The patient was followed up regularly at the Mycetoma Clinic  of the MRC.After 1 month of treatment, the swelling was reduced in size and in 6 months the lesion had disappeared.The lesion ultrasound examination revealed no cavities or grains.The patient continued the treatment for another 3 months.Four lesion site ultrasound examinations conducted 3 months apart showed no evidence of recurrence.

Discussion
Mycetoma is reported globally but is endemic in tropical and subtropical regions in a belt known as the mycetoma belt. 1,2It includes the countries of Sudan, Somalia, Senegal, India, Yemen, Mexico, Venezuela, Columbia, Argentina and a few others. 3,4udan is known to have the highest reported mycetoma prevalence. 5,6The geographical distribution of mycetoma depends on environmental parameters that include temperature, humidity and rainfall. 7,8More than 70 causative agents are responsible for mycetoma and the distribution of these causative agents is not uniform globally. 9Madurella mycetomatis, the most common causative agent of eumycetoma, is reported extensively in Africa and India, but rarely in South America. 10Nocardia brasiliensis is the actinomycetoma causative agent most frequently reported in South America but is rarely seen in Africa. 11t should be noted that A. (A) madurae is the only mycetoma causative agent reported in all the mycetoma-endemic regions. 12ctinomadura commonly resides in the soil, and the members of this genus encompass 40 species. 13Apart from A. madurae, other Actinomadura species have been reported to cause mycetoma.These include Actinomadura pelletieri, Actinomadura Transactions of the Royal Society of Tropical Medicine and Hygiene bangladeshensis and A. mexicana 14 (Table 1), which produce different red and white/yellow grains.
A. mexicana was initially described by Quintana et al. 15 in 2003 following isolation of the organism from the soil in Mexico, hence the name.A. mexicana was not known to cause human mycetoma until 2020, when Bessis et al. 16 reported it for the first time in a 38-year-old woman from the Caribbean region with actinomycetoma.However, mycetoma due to A. mexicana could be more prevalent than currently thought.Macroscopically, A. mexicana grains have white to yellow colour similar to the grains seen in A. madurae and S. somaliensis.In histopathological sections, A. mexicana grains are indistinguishable from those of A. madurae, as they both stain deep blue in H&E-stained sections. 17When cultured, A. mexicana and A. madurae morphology are also comparable.Therefore misidentifications may explain the rarity of this organism.
The reported patient is from Central Sudan, where eumycetoma due to Madurella mycetomatis is predominant and actinomycetoma is less commonly reported. 3,4More in-depth environmental study of the patient's village is needed to explain the occurrence of the reported causative organism.Mycetoma is characterised by a triad of painless subcutaneous swellings, grains and draining sinuses. 18However, the reported patient had no sinuses, which is more commonly seen with actinomycetoma, and the diagnosis was established by lesional ultrasound and histological examinations.The explanation for this presentation is unclear.
The reported patient was treated with an antibiotic combination similar to that reported by Bessis et al., 16 which was 2 g amoxicillin/clavulanate and 1600 mg/280 mg sulfamethoxazole/trimethoprim three times a day.Both patients showed a marked improvement after 3 months of therapy.In our patient, the swelling completely diminished, while in the case presented by Bessis et al., 16 a marked reduction in size and pain was noted.This indicates that the causative organism responds well to a combination of antibiotics.
Of the 1549 actinomycetoma patients seen at the MRC, 576 were due to A. madurae, 175 to A. pelletieri and 798 to Streptomyces species.The cure rate of patients with A. madurae, A. pelletieri and Streptomyces species was 80.6%, 88.9% and 90.5%, respectively.Interestingly, the average treatment duration for A. pelletieri was 16 months and for A. madurae it was 24 months.Both the A. mexicana patients were cured within 6-9 months.This could indicate that mycetoma caused by A. mexicana is more responsive to treatment than the other Actinomadura species.
In conclusion, we report the first patient with actinomycetoma caused by A. mexicana in Sudan.The diagnosis was established by molecular identification of the organism.Hence, molecular identification should be part of the routine diagnostic tools.The patient responded well to medical treatment and in a shorter time and therefore had a better prognosis compared with other actinomycetoma-causative agents.

Figure 1 .
Figure 1.X-ray of the left foot in a lateral view showing no apparent abnormality.

Figure 2 .
Figure 2. Photograph shows the lesion ultrasound examination with thick-walled multiple cavities (red arrow) that contain fluid, with sharp echogenic grains (blue arrows) and intact bone, in line with eumycetoma.

Figure 3 .
Figure 3. (A) Photomicrograph shows A. mexicana grains, which are opaque at the periphery with deep blue staining, the centre is less densely stained and the periphery is slightly eosinophilic (H&E, ×10).(B) A. madurae grains.The periphery is dense, homogeneous and deep blue, while the centre is less dense or even appears hollow (H&E, ×10).

Figure 4 .
Figure 4. (A) Photograph shows A. mexicana colonies with a convex surface and wrinkled in shape in yeast extract agar (YEA).(B) Photomicrograph shows A. madurae colonies after a 10-d incubation, showing a pink to pallid red, convex, wrinkled morphology in YEA.

Table 1 .
Characteristics of Actinomadura species that cause human diseases