Direct 16S/ITS rRNA Gene PCR followed by Sanger Sequencing for Detection of Mycetoma causative Agents in Dakar, Senegal: A pilot study among patients with mycetoma attending Aristide Le Dantec University Hospital

doi:10.21203/rs.3.rs-4455751/v1

Introduction. A mycetoma is defined as “any pathological process in which fungal or actinomycotic agents of exogenous origin produce grains”. A precise identification of the causal agents is critical for the therapeutic outcome. Thus, the objective of this study was to identify the pathogens of mycetoma using direct 16S/ITS rRNA gene polymerase chain reaction (PCR) followed by Sanger sequencing directly on grains.

Materials and Methods. In sum, 32 samples including 15 black grains, 12 red grains, and five white/yellow grains collected from patients with mycetoma at the Aristide Le Dantec University Hospital in Dakar, Senegal, between October 2014 and September 2020 were submitted to PCR/sequencing in IHU Méditerranée Infection in Marseille, France. For black grain eumycetoma, the ITS rRNA region was targeted. Similarly, the 16S rRNA gene was targeted for red grain actinomycetoma. These two regions were targeted in parallel for white/yellow grains, which could be of either bacterial or fungal origin. The obtained sequences were assembled and searched using BLAST against the NCBI GenBank nucleotide database with DNA sequence-based species identification defined by ≥99% sequence similarity.

Results. The age of the patients ranged from 14 to 72 years with a mean age of 36±14 years. Thirteen (86%) of the 15 samples with black grains, were successfully sequenced with only one established eumycetoma pathogen, M. mycetomatisidentified in 11 (73%). Cladosporium sphaerospermum was identified in one sample. For the 16S rRNA sequencing about red grains, a 58.3% (7/12) success rate was obtained with Actinomadura pelletieri identified in six (06) samples. Among the five samples sequenced twice, the ITS rRNA sequencing success rate was 60% (3/5); and no mycetoma causative agent was identified. The 16S rRNA sequencing success rate was 40% (2/5) with the established actinomycetoma causative organism, Actinomadura madurae, identified in one. In the second, A. geliboluensis was identified.

Conclusion. Overall, direct 16S/ITS rRNA sequencing on grains for the detection and identification of mycetoma pathogens was successful in 59.4% of cases. The success rate depended on the colour of the grains. It was 80% for black, 50% for red and 40% for white/yellow grains. Fungi, led by Madurella mycetomatis, were the predominant pathogens identified. We identified two probable new causal agents, namely Cladosporium sphaerospermum, and Actinomadura geliboluensis. Yet, the involvement of both deserves confirmation.

A mycetoma is defined as “any pathological process in which fungal or actinomycotic agents of exogenous origin produce grains”. The originality of this disease is that it can be caused either by fungi (eumycetoma) or by aerobic Actinomycetales (actinomycetoma) [1, 2]. This infectious disease begins as a small nodule, often neglected by the patient, but gradually develops. If not adequately treated, it develops into the characteristic chronic presentation of tumour-like swellings that shed granules [3]. The infection has usually been developing for several years before the first consultation with a specialist. This is a major cause of delay in treatment, with most patients presenting late at an advanced stage of the disease with massive deformities, disability and complications that can be life-threatening [4].

Although it is relatively easy to make a diagnosis of mycetoma in clinical practice, it is impossible to distinguish eumycetoma from actinomycetoma in most cases, while their treatment and prognosis are drastically different [4]. Therefore, the cornerstone of mycetoma diagnosis is the identification of the aetiological agent, which allows the selection of the appropriate treatment. This biological diagnosis is demanding and often unavailable in endemic areas where the specialised structures to carry out this diagnosis are either scarce or non-existent [2, 4]. This is one of the reasons why mycetoma was added to the list of Neglected Tropical Diseases (NTDs) by the World Health Organisation (WHO) in 2013 [5], bringing much more attention to the disease [6].

The mycetoma endemic area includes many tropical and subtropical regions of the so-called mycetoma belt [1]. In Africa, Senegal is one of these regions where mycetoma is endemic. Indeed, it was in Saint-Louis, northern Senegal, that the first African case of mycetoma was reported by Le Dantec in 1894 [4]. Today, more than a century later, the problem of mycetoma remains unresolved in Senegal. Recently, in 2019, a laboratory-based epidemiological study conducted at the Aristide Le Dantec University Hospital Centre in Dakar, one of the main centres for biological diagnosis of mycetoma in the country, reported 338 cases of mycetoma recorded over an 18-year period between 1993 and 2016 [7]. The actual number of cases is probably underestimated, as difficulties in visualising the grains sometimes make diagnosis impossible.

In addition to mycological diagnosis, conventional techniques for the diagnosis of mycetoma include imaging, cytology and histopathology. Mycological diagnosis is generally based on direct microscopic examination and culture. Unfortunately, these phenotypic methods lack sensitivity and usually do not allow species identification [8]. In addition, molecular biology has shown that a species previously identified by phenotypic criteria may be a complex of multiple cryptic species. This is the case for Madurella mycetomatis, the most common cause of mycetoma [3]. It is actually a complex of species including M. fahalii, M. tropicalis and M. pseudomycetomatis [9]. This precise identification is all the more important as these mycetoma pathogens differ in their antifungal susceptibility profiles. Indeed, M. fahalii has been shown to be less susceptible to azoles (especially voriconazole) than other Madurella species [9, 10].

PCR/sequencing has been successfully used to identify mycetoma pathogens, particularly those with black grains [11]. However, to the best of our knowledge, such an approach has not yet been initiated in Senegal, especially directly on grains. Therefore, in order to contribute to the epidemiology of mycetoma in Senegal, the aim of this study was to identify the causal agents of mycetoma using direct 16S/ITS rRNA gene polymerase chain reaction (PCR) followed by Sanger sequencing directly on grains.

Clinical specimens. Clinical samples consisted of grains. They were collected from patients who were diagnosed with mycetoma after a dermatological consultation and who visited the parasitology and mycology laboratory at the Aristide Le Dantec University Hospital in Dakar, Senegal, between October 2014 and September 2020. These patients principally came from different regions of Senegal.

The grains were collected using a scalpel and a swab and placed directly into a tube containing sterile distilled water. As many grains as possible were collected and divided into two parts. One part was used for conventional diagnosis in Dakar and the other, which is the subject of this retrospective evaluation, was later used for PCR/sequencing in Marseille, France. Part of this latter sample, intended for molecular biology, was also used to repeat the culture in cases where the former part was contaminated at an early stage. For retrospective evaluation, samples with remaining grains were selected for PCR/sequencing.

DNA Extraction. First, the grains were placed in 2 mL tubes containing ceramic beads and 600 µL of Lysis Buffer G2 (provided with the Qiagen^TM Tissue Kit) for mechanical lysis at 6 m/s for 40 s using a FastPrep-24™ instrument, followed by centrifugation at 10,000 x g for 1 min. DNA extraction was then performed using a NucliSENS^® easyMAG^® instrument (bioMérieux, France). Finally, a total elution volume of 100 µL of genomic DNA was extracted and stored at -20 °C for further analysis.

PCR followed by Sanger Sequencing. For black grain eumycetoma, the ITS rRNA region was targeted. Similarly, the 16S rRNA gene was targeted for red grain actinomycetoma. These two regions were targeted in parallel for white/yellow grains, which could be of either bacterial or fungal origin. These genes were amplified with specific primers (Table 1).

Table 1. Sequences of the primers used in this study.

The standard PCR mix was prepared using the AmpliTaq Gold^TM 360 Master Mix Kit (Applied Biosystems^®) for a final volume of 25 μL: 12.5 μL buffer, 6 μL DNase/RNase-free water, 0.75 μL primers (forward and reverse) and 5 μL extracted DNA. All PCRs were performed with an initial denaturation temperature of 95 °C for 15 min, followed by 40 cycles of 95 °C for 30 s, a specific hybridisation temperature depending on the primer system for 30 s, an extension temperature of 72 °C for a time depending on the number of base pairs of each targeted DNA sequence, and a final extension of 72 °C for 5 min and storage at 4 °C. The amplicons were resolved on a 2 % agarose gel, stained with ethidium bromide and visualised under a UV light source (E-gel^TM Imager). Prior to sequencing, the amplicons were purified using the NucleoFastH 96 PCR kit (Machery-Nagel, France). Sequencing was performed by the BigDye reaction using the MiniAmp thermocycler (Thermo Fisher Scientific, USA) and the same primers as above. The mix for this reaction contained 3.5 μL water, 1 μL BigDye, 1 μL BigDye buffer, 0.5 μL primers (reverse and forward), 4 μL purified DNA for a total volume of 10 μL. The following programme was used for all BigDye reactions 96 °C for 1 min, then 25 cycles of 96 °C for 10 s, 50 °C for 30 s and 60 °C for 3 min with storage at 15 °C. BigDye sequencing products were purified using Sephadex^TM G50 DNA purification. Sequencing reactions were run on a 3500 XL Genetic Analyzer (Applied Biosystems, Inc.). Sequences were assembled with Chromas pro version 1.7.7 (Technelysium Pty Ltd). Sequences were searched using BLAST (Basic Local Alignment Search Tool at https://blast.ncbi.nlm.nih.gov/Blast.cgi) against the NCBI GenBank nucleotide database. DNA sequence-based species identification was defined by ≥99% sequence similarity.

Characteristics of the population. We included 32 patients who had been diagnosed with mycetoma at the Parasitology and Mycology Laboratory of Aristide Le Dantec University Hospital in Dakar, Senegal. One sample per patient, where grains were visible, was analysed. The age of the patients ranged from 14 to 72 years. The mean age was 36±14 years. The characteristics of these patients are summarised in Table 2. Of the 32 samples analysed, 15 (47%) contained black grains, 12 (37.5%) red grains and the remainder white/yellowish grains.

Table 2. Sociodemographic and clinical characteristics of the study population

Characteristics	Total (n)	Percentage (%)
Sex
Female	7	22
Male	25	78
Age (years)
<20	3	9.4
20-39	18	56.3
40-59	10	31.3
≥ 60	1	3
Region of origin (geographic location)
Dakar (Extreme West)	1	3.1
Diourbel (West)	6	18.8
Fatick (West)	4	12.5
Kaolack (Mid-West)	1	3.1
Louga (Northwest)	4	12.5
Matam (Northeast)	3	9.4
Thiès (West)	8	25
*Country bordering Senegal	5	15.6
^§Occupation
Farmer	8	25
Livestock breeder	2	6.2
Farmer/breeder	3	9.4
Driver	1	3.1
Koranic teacher	1	3.1
Koranic student	1	3.1
Builder	2	6.2
Trader	1	3.1
Household	3	9.4
Mycetoma lesion site
Foot	22	68.8
Knee	3	9.3
Thigh	1	3.1
Buttock	2	6.3
Perineum/bladder	2	6.3
Back	1	3.1
Forehead	1	3.1
*Four cases from Mauritania and one from Cape Verde. ^§The occupation was not specified for the rest of the patients.

ITS rRNA sequencing diagnosis of black grains mycetoma. Thirteen (86%) of the 15 samples with black grains, were successfully sequenced. Only one established eumycetoma pathogen, M. mycetomatis, was identified in 11 (73%). Cladosporium sphaerospermum was identified in one sample (Table 3).

Table 3. ITS rRNA sequencing results of (n = 15) black grains Eumycetoma.

Sample ID		Identified fungi	Identity*	Clinical significance	GenBank Accession No
14/M13	Madurella mycetomatis		100%	Significant	PP506153
14/M14	Cladosporium sphaerospermum		99.05%	New agent?	PP506154
15/M02	Madurella mycetomatis		100%	Significant	PP506155
15/M07	Candida tropicalis		99.78%	Contamination	-
15/M12	Madurella mycetomatis		100%	Significant	PP506156
16/M13	Madurella mycetomatis		99.32%	Significant	PP506157
16/M19	Madurella mycetomatis		100%	Significant	PP506158
16/M20	None		-	Failure	-
17/M01	None		-	Failure	-
19/M10	Madurella mycetomatis		99.27%	Significant	PP506159
19/M19	Madurella mycetomatis		100%	Significant	PP506160
20/M01	Madurella mycetomatis		100%	Significant	PP506161
20/M13	Madurella mycetomatis		99.26%	Significant	PP506162
20/M14	Madurella mycetomatis		100%	Significant	PP506163
20/M15	Madurella mycetomatis		99.81%	Significant	PP506164

* Percentage of nucleotide sequence identify with the sequence in the National Centre for Biotechnology Information GenBank nucleotide database.

16S rRNA sequencing diagnosis of red grains mycetoma. For the 16S rRNA sequencing about red grains, a 58.3% (7/12) success rate was obtained. Actinomadura pelletieri was identified in six (06) samples (Table 4).

Table 4. 16S rRNA sequencing results of (n = 12) red grains actinomycetoma.

Sample ID	Identified bacteria	Identity*	Clinical significance	GenBank Accession No
15/M04	Actinomadura pelletieri	99.43%	Significant	PP503340
15/M08	Actinomadura pelletieri	99.38%	Significant	PP503341
16/M03	Actinomadura pelletieri	99.13%	Significant	PP503342
16/M08	Aureimonas altamirensis	99.52%	Contamination	-
16/M14	Actinomadura pelletieri	99.80%	Significant	PP503343
16/M26	Actinomadura pelletieri	97.85%	Significant	PP503344
17/M02	None	-	Failure	-
19/M05	Actinomadura pelletieri	99.32%	Significant	PP503345
19/M08	None	-	Failure	-
19/M17	None	-	Failure	-
19/M18	None	-	Failure	-
19/M20	None	-	Failure	-

* Percentage of nucleotide sequence identify with the sequence in the National Centre for Biotechnology Information GenBank nucleotide database.

Combined 16S and ITS rRNA sequencing diagnosis of white/yellowish grains mycetoma. Five samples were sequenced twice, by successively targeting 16S rRNA and ITS rRNA. The ITS rRNA sequencing success rate was 60% (3/5); and no mycetoma causative agent was identified. The 16S rRNA sequencing success rate was 40% (2/5). The established actinomycetoma causative organism, Actinomadura madurae, was identified in one. In the second, A. geliboluensis was identified (Table 5).

Table 5. 16S and ITS rRNA sequencing results of (n = 5) white/yellow grains mycetoma.

Sample ID	Identified fungi		Clinical significance	Accession numbers (GenBank)
Sample ID	ITS rRNA sequencing	16S rRNA sequencing	Clinical significance
	(NCBI Similarity)
16/M01	None (NA)	Actinomadura madurae (99.02%)	Significant	PP503346
16/M12	None (NA)	Actinomadura geliboluensis (100%)	New agent?	PP503347
20/M02	Candida tropicalis (100%)	None (NA)	Contamination	-
20/M03	Aspergillus fumigatus (97.8%)	None (NA)	Contamination	-
20/M18	Aspergillus fumigatus (100%)	None (NA)	Contamination	-
NA: Not Applicable

This retrospective clinical evaluation study of patients diagnosed with mycetoma, who attended the Parasitology and Mycology laboratory at Aristide Le Dantec University Hospital in Dakar identified the causative agents of mycetoma in Senegal using direct 16S/ITS rRNA gene PCR followed by Sanger sequencing directly from grains. By using conventional macroscopic mycetoma diagnosis, we identified 47% black grain eumycetoma, and 38% red grain actinomycetoma. These findings are in line with the latest mycetoma epidemiological data in Senegal, which showed prevalences of eumycetoma ranging from 47–70% [6, 7, 12]. The limitation of conventional diagnosis rationalises the use of DNA-based diagnostic methods. We used direct 16S/ITS rRNA Sanger sequencing from the mycetoma grains to identify the causal agents. With this approach, we succeeded in 80% for black grains mycetoma diagnosis, and identified predominantly M. mycetomatis. This relatively high success rate might be explained by the large quantity of grains produced by this species, which allows us to obtain a sufficient quantity of fungal DNA and limits the amplification of contaminant microorganisms that often remain in samples, despite previous washing. These results are consistent with a similar study carried out by Ahmed et al. [16] in Sudan, where M. mycetomatis was involved in 90% black grains mycetoma. Our results also confirmed the study by K et al. [17], which demonstrated the feasibility and reliability of direct DNA-sequencing for the diagnosis of eumycetomas, especially for the identification of M. mycetomatis. In contrast, the implication of Cladosporium sphaerospermum as a causal agent of black grains eumycetoma remains controversial. Unfortunately, the retrospective study design hinders further investigation. The low number of grains produced by other pathogens explains the lower success rate for red and white/yellowish grains mycetoma. With regard to actinomycetes, Actinomadura pelletieri was the predominant pathogen (6/12), and the second most frequent pathogen in our study. In addition, a new species, A. geliboluensis was identified among white/yellowish grains mycetoma. To our knowledge, this is the first time that this species has been implicated in white/yellow grain mycetoma. In fact, this species was first isolated from soil, described and characterised by Turkish authors in 2012 [18]. Therefore, further investigations on this agent deserve to be carried out, although they may be compromised for the reasons mentioned above. The aetiological agent remained unknown in 40.6% of mycetoma cases, despite both 16S and ITS rRNA sequencing. In fact, grains contain a "microbiome" and DNA-based identification will only identify the microorganism whose DNA is most amplified. Otherwise, Sanger sequencing will generate mixed sequences that compromise DNA-based identification. This hypothesis is in line with the 16S rRNA deep sequencing data of Salipante et al., [19] which allowed the identification of Actinomadura madurae as the pathogen involved in a mycetoma where conventional diagnosis by culture or Sanger sequencing had always been interpreted as contamination. The authors concluded that the proliferation of contaminant micro-organisms limits diagnosis by culture and direct Sanger sequencing [19].

The mean age of our patients was similar to those described in a previous study carried out in the same laboratory between 2008 and 2010 by Ndiaye et al, [12]. Similar results were found in studies carried in the countries bordering Senegal, including in Mauritania [13] and in Mali [14]. This relatively young age of the patients could be explained by their strong involvement in rural activities such as agriculture and livestock breeding. These activities being more practiced by male than women, this could justify the predominance of men in our study population. This observation is shared in almost all studies because the exercise of these activities exposes one to inoculating trauma, particularly through thorn pricks and minor injuries [1]. Our study was no exception to this rule since most of the patients included were farmers (25%) and/or livestock breeders. The characteristics of our study population were rather typical [15].

The majority of our patients came from the west (region of Thiès, Diourbel and Fatick) and the north (region of Louga and Matam) of Senegal. This finding is consistent with a previous study in Senegal [12] and may be explained by the hot and dry climate in these regions, which favours the development of causative microorganisms. One case was reported from the region of Dakar (the capital city). Mycetoma is less common in this region. This could indicate a change in the epidemiology of mycetomas in Senegal [7]. However, these data should be interpreted with caution. In fact, patients know that Dakar is the only place in Senegal where medical mycology laboratories exist, and they often prefer to give their visiting address in Dakar rather than their home address.

Consistent with our findings, a predominance of podal location of mycetoma has been observed in the majority of previous studies [3, 12, 15]. Involvement of the knee and thigh also occurred. The high exposure to inoculation trauma may explain this predominance of lower limb involvement. After the lower limbs, the gluteal and perineal areas were the second most common, which may be related to daily rural living habits, especially sitting on the floor and sometimes lying on their backs.

Overall, direct 16S/ITS rRNA sequencing on grains for the detection and identification of mycetoma pathogens was successful in 59.4% of cases. The success rate depended on the colour of the grains. It was 80% for black, 50% for red and 40% for white/yellow grains. Fungi, led by Madurella mycetomatis, were the predominant pathogens identified. We identified two probable new causal agents, namely Cladosporium sphaerospermum, involved in black grains mycetoma, and Actinomadura geliboluensis, involved in white/yellowish grains mycetoma. Yet, the involvement of both deserves confirmation.

Conflicts of Interest

The authors have no conflict of interest to declare regarding this study.

Funding

The authors received no specific funding for this study.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Authors’ Contributions

Khadim Diongue and Stéphane Ranque were responsible for the study design; Khadim Diongue, Jihane Kabtani, Jean-Noel Dione and Stéphane Ranque were involved in development and methodology; data analysis and interpretation was performed by Khadim Diongue, Abdoulaye Diop, and Stéphane Ranque; Khadim Diongue wrote the manuscript; and Mamadou Alpha Diallo, Coralie L’Ollivier, Mame Cheikh Seck, Mouhamadou Ndiaye, Aida Sadikh Badiane and Daouda Ndiaye revised the manuscript. All the authors have read and agreed to the final version of the manuscript.

Acknowledgments

This work was performed as part of the collaboration between the Cheikh Anta Diop University of Dakar, Senegal, and the “Méditerranée Infection” institute of Marseille, France.

Ethical Approval

This study was approved by the National Committee of Ethics for Health Research of Senegal (CNERS/0212/2016/CER/UCAD) and in accordance with the procedures established by the University Cheikh Anta DIOP of Dakar (UCAD) for the ethical approval of any research involving human participants. Informed consent was obtained from the study participants.

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Direct 16S/ITS rRNA Gene PCR followed by Sanger Sequencing for Detection of Mycetoma causative Agents in Dakar, Senegal: A pilot study among patients with mycetoma attending Aristide Le Dantec University Hospital

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Abstract

Introduction

Materials and methods

Results

Discussion

Conclusion

Declarations

References

Status:

Journal Publication

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