Association of Toll-like Receptors 1, 2, 4, 6, 8, 9 and 10 Genes Polymorphisms and Susceptibility to Pulmonary Tuberculosis in Sudanese Patients.

Background: Genetic factors are important contributors to the development of a wide range of complex disease. Polymorphisms in genes encoding for toll-like receptors (TLRs) usually influence the efficiency of the immune response to infection and are associated with disease susceptibility and progression. Therefore, we aim to describe the first association between TLR1, TLR2, TLR4 TLR6, TLR8, TLR9 and TLR10 genes polymorphisms and susceptibility to pulmonary tuberculosis (PTB) in Sudanese patients. Methodology: Here we performed a case study which included 160 tuberculosis patients and 220 healthy matched controls from Sudan. In the study population, we evaluated the possible association between 86 markers in TLR1, TLR2, TLR4 TLR6, TLR8, TLR9 and TLR10 genes polymorphisms and susceptibility to PTB disease in Sudanese population using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Results: From our results it appeared that in the PTB population the TLR1 (rs5743557, rs4833095, rs5743596), TLR2 (rs5743704, rs5743708, rs3804099), TLR4 (rs4986790, rs4986791), TLR6 (rs5743810), TLR8 (rs3764879, rs3764880), TLR9 (rs352165, rs352167, rs187084) and TLR10 (rs4129009) were significantly more often encountered (p<0.0001) than in the control population and were associated with PTB in the Sudanese population. For the other polymorphisms tested, no association with PTB was found in the population tested. Conclusion: The work describes novel mutations in TLR1, TLR2, TLR4, TLR6, TLR8, TLR9 and TLR10 genes and their association with PTB infection in Sudanese population. These results will enhance our ability to determine the risk of developing the disease by targeting specific TLR pathways to reduce the severity of the disease. Future studies are needed in a larger sample to replicate our findings and understand the mechanism of association of TLR polymorphism in PTB.

Wako ß-D-glucan assay can be used to measure serum ß-D-glucan in Sudanese patients to aid with diagnosis of eumycetoma caused by Madurella mycetomatis.

BACKGROUND: Eumycetoma is a neglected tropical infection of the subcutaneous tissue commonly caused by the fungus Madurella mycetomatis. Previously, we demonstrated that ß-D-glucan was present in the serum of eumycetoma patients. OBJECTIVE: To compare the performance of the recently approved easy-to-use Wako ß-D-glucan assay to that of the Fungitell assay in eumycetoma patients. METHODS: Using sera obtained from 41 eumycetoma, 12 actinomycetoma and 29 healthy endemic controls, we measured the ß-glucan serum concentrations using the Wako assay and compared the performance to that of the Fungitell assay. RESULTS: With the Fungitell assay, median ß-glucan serum concentrations of 208, 70 and 27 pg/ml were obtained for the 41 eumycetoma patients, the 12 actinomycetoma patients and the 29 healthy endemic controls, respectively. With the Wako assay these concentrations were 14.45, 11.57 and 2.5 pg/ml, respectively. We demonstrated that when using the optimized cut-off value (5.5 pg/ml) for the Wako assay, the Wako and Fungitell assays had comparable performance in terms of sensitivity and specificity. CONCLUSION: The Wako assay is comparable to the Fungitell assay for measurement of serum ß-glucan in mycetoma patients and hence can be used in combination with current diagnostic tools. However, this test should be used in combination with other tests to differentiate actinomycetoma from eumycetoma.

Metagenomic detection of eumycetoma causative agents from households of patients residing in two Sudanese endemic villages in White Nile State.

Eumycetoma is a chronic debilitating fungal disease endemic to tropical and subtropical regions, with Sudan featuring the highest eumycetoma incidence. Among the 50 species of fungi most commonly associated with eumycetoma Madurella mycetomatis (M. mycetomatis) is often referenced as the most common pathogen. However, there is an enormous knowledge gap related to this neglected disease and its pathogenesis, epidemiological features, and host-specific factors that could contribute to either the host susceptibility and resistance. In this study, we were able to utilize a metagenomic approach and samples collected from clinical black grains (BG) and familiar household environments aimed to assay both the habitat of eumycetoma-associated fungi and its possible connection with eumycetoma patients living in two different eumycetoma endemic villages within the White Nile State of Sudan. DNA sequencing targeting the fungal ITS2 domain was performed on soil, animal dung, housing walls and roofs, and Acacia-species thorn samples and compared with culture-dependent methods of fungal isolation. Additionally, we compared the soil samples obtained in the endemic zone with that from non-endemic zones, including Wagga village in Kassala State and Port Sudan suburb in Port Sudan State. Overall, a total of 392 Amplicon Sequence Variants (ASVs) were detected by ITS2 metagenomics Eumycetoma causative organisms accounted for 10% of total ASVs which included 11 genera: Exserohilum (2%), Aspergillus (1.7%), Curvularia (1%), Alternaria (0.9%), Madurella (0.5%), Fusarium (0.4%), Cladosporium (0.2%) Exophiala (0.15%), and, in a lesser extent, Microascus (0.05%) Bipolaris and Acremonium (0.01%) for each. Only five genera were identified by culture method, which included Fusarium (29%), Aspergillus (28%), Alternaria (2.5%), Bipolaris (1.6%), and Chaetomium (0.8%). M. mycetomatis was detected within all the studied patients' houses, accounting for 0.7% of total sequences. It was the first common eumycetoma-associated agent detected in soil samples and the third common in the dung and wall samples. In contrast, it was not detected in the roof or thorn samples nor in the soils from non-endemic regions. Exserohilum rostratum, Aspergillus spp and Cladosporium spp were detected in all samples. M. mycetomatis and other eumycetoma-associated fungal identified in the patients' black grains (BG) samples by metagenomics were identified in the environmental samples. Only Acremonium alternatum and Falciformispora senegalensis, responsible for eumycetoma in two patients were not detected, suggesting the infections in these patients happened outside these endemic areas. The soil, animal dung, and houses built from the same soil and dung are the main risk factors for M. mycetomatis infection in these endemic villages. Furthermore, the poor hygienic and environmental conditions, walking barefooted, and the presence of animals within the houses increase the risk of M. mycetomatis and other fungi causing eumycetoma.

A possible role for ticks in the transmission of Madurella mycetomatis in a mycetoma-endemic village in Sudan.

BACKGROUND: Currently there is a wide knowledge gap in our understanding of mycetoma epidemiological characteristics, including the infection route. METHODS: A cross-sectional descriptive epidemiological study was carried out to determine the role of exposure to animals and insects such as ticks in the transmission of eumycetoma in two adjacent villages at eastern Sudan. RESULTS: Significant differences were found between the two villages in the level of contact and exposure to animals and ticks, the percentages of people bitten by ticks, participation in cleaning animal pens and knowledge of the medical importance of ticks. In the village with a high mycetoma prevalence rate, there were high infestation rates of ticks in domestic animals. Hyalomma and Rhipicephalus species were the most prevalent species in houses with mycetoma patients and together they constituted 83% of the total collection. Pool screening of vectors for the detection of Madurella mycetomatis recombinant RNA genes showed one positive pool from Rhipicephalus evertsi following amplification of the universal fungal primer and one positive sample from Hyalomma rufipes following the use of a specific primer. CONCLUSION: The findings indicate a possible role of ticks in the transmission of eumycetoma causative agents. However, further in-depth studies are needed to verify this.

Metagenomics of black grains: new highlights in the understanding of eumycetoma.

BACKGROUND: Eumycetoma is a chronic subcutaneous granulomatous disease that is endemic in Sudan and other countries. It can be caused by eight different fungal orders. The gold standard diagnostic test is culture, however, culture-independent methods such as imaging, histopathological and molecular techniques can support diagnosis, especially in cases of negative cultures. METHODS: The amplicon-based internal transcribed spacer 2 metagenomic technique was used to study black grains isolated from 14 tissue biopsies from patients with mycetoma. Furthermore, mycological culture and surgical biopsy histopathological examinations of grains were performed. RESULTS: Madurella mycetomatis (n=5) and Falciformispora spp. (n=4) organisms were identified by culture and confirmed by metagenomics. Metagenomics recognised, at the species level, Falciformispora as Falciformispora tompkinsii (n=3) and Falciformispora senegalensis (n=1), while in culture-negative cases (n=5), Madurella mycetomatis (n=3), Falciformispora senegalensis (n=1) and Fusarium spp. (n=1) were identified. Interestingly, the metagenomics results showed a 'consortium' of different fungi in each sample, mainly Ascomycota phylum, including various species associated with eumycetoma. The microbial co-occurrence in eumycetoma showed the co-presence of Madurella with Trichoderma, Chaetomium, Malasseziales and Sordariales spp., while Falciformispora co-presented with Inocybe and Alternaria and was in mutual exclusion with Subramaniula, Aspergillus and Trichothecium. CONCLUSION: Metagenomics provides new insights into the aetiology of eumycetoma in samples with negative culture and into the diversity and complexity of grains mycobiota, calling into question the accuracy of traditional culture for the identification of causative agents.

Multiple extensive Madurella mycetomatis eumycetoma lesions: a case report and review of the literature.

In this communication, we report on the first-ever patient presenting with 17 lesions of Madurella mycetomatis eumycetoma in different parts of the body. The reported patient is a 15-y-old female who presented to the Mycetoma Research Centre, University of Khartoum, Khartoum, Sudan, in 2017 with recurrent back and anterior abdominal wall eumycetoma lesions. They were surgically excised, and during the course of follow-up, she developed 15 other eumycetoma lesions, scattered mostly on her upper and lower limbs and gluteal region. The diagnosis of Madurella mycetomatis was confirmed by molecular identification of grains and culture, histopathological examination and ultrasound examination. The cause and the explanation of such a presentation in puzzling; it is not due to multiple subcutaneous implantations because of the lack of history of relevant local trauma. Lymphatic spread is unlikely as the distribution of the lesions is not in line with this. Haematogenous spread is a possible explanation. This rare presentation of eumycetoma poses a great challenge for diagnosis and management.

Mycetoma caused by Microascus gracilis: a novel agent of human eumycetoma in Sudan.

Species of the genus Microascus are uncommon agents of human diseases despite their ubiquitous presence in the environment. In this communication, the first case of white grain eumycetoma caused by the fungus Microascus gracilis is reported. The patient was initially misdiagnosed as having actinomycetoma based on the grains morphological and cytological features and was treated with antimicrobial therapy with no clinical improvement. She underwent wide local surgical excision to improve the response to medical treatment and further grain cultural, molecular and taxonomy techniques were conducted and the diagnosis of mycetoma due to M. gracilis was established. The antifungal susceptibilities of this isolate to nine drugs were tested in vitro and they showed poor activity. Combination therapy with surgery and itraconazole led to complete recovery. A medical literature search revealed no previous report on M. gracilis as a causative agent of eumycetoma and hence we are reporting this new causative agent of human eumycetoma. Also, the difficulty in the management of this patient emphasizes the need for accurate and appropriate diagnostic tests for the identification of mycetoma-causative organisms and thus proper management.

Prevalence of Helicobacter pylori among Sudanese patients diagnosed with colon polyps and colon cancer using immunohistochemistry technique.

OBJECTIVES: Infection with the bacteria Helicobacter pylori has been classified as class one carcinogen associated with increasing susceptibility of gastritis and gastric carcinoma. This study is aiming at investigating the prevalence of H. pylori among colon polyps and colon cancer patients. A descriptive cross-sectional hospital-based study was conducted between February and June 2017. Sixty-nine formalin-fixed paraffin blocks collected from colon polyps and colon cancer patients to detect H. pylori using immunohistochemistry technique. RESULTS: Of the 69 patients included in the study, 39 (56.5%) males and 30 (43.5%) were females, their age ranged from 21 to 80 years with a mean age of 47.1 ± 19.7. Of the 69 colon polyps and colon cancer patients, 44 (63.8%) were diagnosed as adenocarcinoma, 10 (14.5%) colitis, 15 (21.7%) juvenile polyposis syndrome. The results of immunohistochemistry technique showed the presence of 16 (23.2%) positive patients for H. pylori infection. Of these 16, 13 (81.3%) patients were diagnosed with adenocarcinoma and 3 (18.7%) patients were diagnosed with juvenile polyps. The results of H. pylori detection among the different colon polyps and colon cancer patients were showing a statistically significant association for H. pylori infection and adenocarcinoma, P value 0.028.

Chaetomium atrobrunneum causing human eumycetoma: The first report.

In this communication, a case of black grain eumycetoma produced by the fungus C. atrobrunneum is reported. The patient was initially misdiagnosed with M. mycetomatis eumycetoma based on the grains' morphological and cytological features. However, further aerobic culture of the black grains generated a melanised fungus identified as C. atrobrunneum by conventional morphological methods and by internal transcribed spacer 2 (ITS2) ribosomal RNA gene sequencing. This is the first-ever report of C. atrobrunneum as a eumycetoma-causative organism of black grain eumycetoma. It is essential that the causative organism is identified to the species level, as this is important for proper patient management and to predict treatment outcome and prognosis.

Madurella mycetomatis causing multiple mycetoma lesions: a rareclinical presentation

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Application of Isothermal Amplification Techniques for Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma.

Appropriate diagnosis and treatment of eumycetoma may vary significantly depending on the causative agent. To date, the most common fungus causing mycetoma worldwide is Madurella mycetomatis. This species fails to express any recognizable morphological characteristics, and reliable identification can therefore only be achieved with the application of molecular techniques. Recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) are proposed as alternatives to phenotypic methods. Species-specific primers were developed to target the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region of M. mycetomatis. Both isothermal amplification techniques showed high specificity and sufficient sensitivity to amplify fungal DNA and proved to be appropriate for detection of M. mycetomatis. Diagnostic performance of the techniques was assessed in comparison to conventional PCR using biopsy specimens from eumycetoma patients. RPA is reliable and easy to operate and has the potential to be implemented in areas where mycetoma is endemic. The techniques may be expanded to detect fungal DNA from environmental samples.

The combination of amoxicillin-clavulanic acid and ketoconazole in the treatment of Madurella mycetomatis eumycetoma and Staphylococcus aureus co-infection.

Eumycetoma is a chronic progressive disabling and destructive inflammatory disease which is commonly caused by the fungus Madurella mycetomatis. It is characterized by the formation of multiple discharging sinuses. It is usually treated by antifungal agents but it is assumed that the therapeutic efficiency of these agents is reduced by the co-existence of Staphylococcus aureus co-infection developing in these sinuses. This prospective study was conducted to investigate the safety, efficacy and clinical outcome of combined antibiotic and antifungal therapy in eumycetoma patients with superimposed Staphylococcus aureus infection. The study enrolled 337 patients with confirmed M. mycetomatis eumycetoma and S. aureus co-infection. Patients were allocated into three groups; 142 patients received amoxicillin-clavulanic acid and ketoconazole, 93 patients received ciprofloxacin and ketoconazole and 102 patients received ketoconazole only. The study showed that, patients who received amoxicillin-clavulanic acid and ketoconazole treatment had an overall better clinical outcome compared to those who had combined ciprofloxacin and ketoconazole or to those who received ketoconazole only. In this study, 60.6% of the combined amoxicillin-clavulanic acid/ketoconazole group showed complete or partial clinical response to treatment compared to 30.1% in the ciprofloxacin/ketoconazole group and 36.3% in the ketoconazole only group. The study also showed that 64.5% of the patients in the ciprofloxacin/ketoconazole group and 59.8% in the ketoconazole only group had progressive disease and poor outcome. This study showed that the combination of amoxicillin-clavulanic acid and ketoconazole treatment is safe and offers good clinical outcome and it is therefore recommended to treat eumycetoma patients with Staphylococcus aureus co-infection.

The association between the interleukin-10 cytokine and CC chemokine ligand 5 polymorphisms and mycetoma granuloma formation.

Mycetoma is a progressive and destructive chronic granulomatous subcutaneous inflammatory disease caused by bacteria and fungi. The genetic determinants for susceptibility to and the development of mycetoma are unclear. Polymorphisms in genes encoding for cytokines and chemokines usually influence the efficiency of the immune response to infection and are associated with disease susceptibility and progression. Therefore, we hypothesized that polymorphisms of CC chemokine ligand 5 (CCL5) and interleukin-10 (IL-10) promoter regions might contribute to the initiation, susceptibility, and severity of eumycetoma. This case-control study included 149 mycetoma patients and 206 healthy matched controls. In the study population, three functional single nucleotide polymorphisms (SNPs) in CCL5 and two in IL-10 were genotyped using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Significant differences in allele distribution were demonstrated for CCL5 -28 C/G (P < 0.0001), CCL5 In1.1 T/C (P < 0.0001) and IL-10 -592 A/C. Since in previous studies it was demonstrated that the genotypes obtained for CCL5 and IL-10 were connected with CCL5 and IL-10 production we measured the serum levels of CCL5 and IL-10 in mycetoma patients and healthy controls. Elevated serum levels for both CCL5 and IL-10 were found in mycetoma patients and we describe that genetic differences in CCL5 and IL-10 are associated with the development of the mycetoma granuloma.

Pleurostomophora ochracea, a novel agent of human eumycetoma with yellow grains.

The first yellow-grain fungal mycetoma, in a 60-year-old man from Central Sudan, is reported. Morphological and phylogenetic analysis of the ribosomal small subunit (SSU), large subunit (LSU), internal transcribed spacer (ITS), ß-tubulin (BT2), actin (ACT1), and elongation factor (TEF1) genes revealed that the isolate deviated from any known agent of mycetoma; it clustered in the genus Pleurostoma (anamorph genus, Pleurostomophora) in the order Calosphaeriales. The novel species, here named Pleurostomophora ochracea, is characterized by phenotypic features. The species proved to be highly susceptible to itraconazole, ketoconazole, posaconazole, and voriconazole, but not to fluconazole. The fungus was inhibited by caspofungin at 8 µg/ml, while no inhibition was found with 5-flucytosine (MIC > 64 µg/ml). Compared to other members of the genus Pleurostomophora, P. ochracea is slow growing, with a relatively high optimum growth temperature (36 to 37°C). This is the first case of a yellow-grain fungal mycetoma; yellow grains are otherwise of bacterial nature. Our case emphasizes that identification of mycetoma agents by the color of the grain only is not sufficient and may lead to inappropriate therapy.