Using a Madurella mycetomatis-specific PCR on grains obtained via non-invasive fine-needle aspirated material is more accurate than cytology.

BACKGROUND: Eumycetoma is a chronic subcutaneous inflammatory fungal infection most often caused by the fungus Madurella mycetomatis. Using a species-specific PCR on DNA directly isolated from grains is currently the most reliable method for species identification. However, so far, PCR has been performed on grains obtained through deep-seated surgical biopsies, which are invasive procedures. Grains can also be obtained via ultrasound-guided fine-needle aspiration (US-FNA). Here we determined the diagnostic performance of species-specific PCRs performed on samples obtained through US-FNA. METHODS: From 63 patients, US-FNA was performed to obtain eumycetoma grains; 34 patients also underwent a deep-seated biopsy. From the grains, DNA was isolated, and one pan-fungal and two M. mycetomatis-specific PCRs were performed. The sensitivity and specificity were determined. RESULTS: Of the 63 patients who underwent US-FNA, 78% (49/63) had evidence of eumycetoma based on cytology and 93.7% (59/63) based on species-specific PCRs. In the 34 patients for whom surgical biopsies were performed as well, 31 patients had a positive PCR for M. mycetomatis when DNA was isolated from the deep-seated biopsy, and 30 had a positive PCR when DNA was obtained from the US-FNA material. This resulted in a 96.8% sensitivity, and 100% specificity with 97.1% diagnostic accuracy for PCR performed on US-FNA. CONCLUSION: PCR performed on the US-FNA material has a similar sensitivity and specificity as PCR performed on deep-seated biopsies. Therefore, when using PCR, a deep-seated biopsy may not be necessary to obtain grains.

Comparing the performance of the common used eumycetoma diagnostic tests.

OBJECTIVES: Mycetoma is a neglected tropical implantation disease caused by 70 different infectious agents. Identifying the causative organism to the species level is essential for appropriate patient management. Ultrasound, histopathology, culture and two species-specific PCRs are most the commonly used methods for species identification in endemic regions. The aim of this study was to compare the diagnostic performance of these commonly used assays using sequencing of barcoding genes as the gold standard. METHODS: This descriptive cross-sectional study was conducted at the Mycetoma Research Centre, University of Khartoum, Sudan. It included 222 patients suspected of fungal mycetoma caused by Madurella mycetomatis. RESULTS: 154 (69.3%) were correctly identified by ultrasound, histology, culture and both species-specific PCRs. In 60 patients, at least one of the diagnostic tests failed to identify M. mycetomatis. Five patients had no evidence of eumycetoma, and for three, only the ultrasound was indicative of mycetoma. The two species-specific PCRs were the most sensitive and specific methods, followed by culture and histology. Ultrasound was the least specific as it only allowed differentiation between actinomycetoma and eumycetoma. The time to result was 9.38 minutes for ultrasound, 3.76 hours for PCR, 8.5 days for histopathology and 21 days for grain culturing. CONCLUSION: Currently, PCR directly on DNA isolated from grains is the most rapid and reliable diagnostic tool to identify M. mycetomatis eumycetoma.

Hepatitis E Virus Outbreak among Tigray War Refugees from Ethiopia, Sudan.

We report hepatitis E virus (HEV) outbreaks among refugees from Ethiopia in Sudan during June 2021-February 2022. We identified 1,589 cases of acute jaundice syndrome and used PCR to confirm HEV infection in 64% of cases. Implementing vaccination, water, sanitation, and hygiene programs might reduce HEV outbreak risk.

Blood meal profile and positivity rate with malaria parasites among different malaria vectors in Sudan.

BACKGROUND: Malaria is a life-threatening public health problem globally with particularly heavy burden in the sub-Saharan Africa including Sudan. The understanding of feeding preference of malaria vectors on different hosts is a major challenge for hindering the transmission cycle of malaria. In this study, blood meals taken by blood-fed Anopheles mosquitoes collected from the field in malaria endemic areas of Sudan were analysed for source of blood meal and malaria parasite presence. METHODS: Anopheles mosquitoes were collected from different regions in Sudan: Khartoum state, Sennar state, Northern state, and El Gedarif state between September 2020 and February 2021. Anopheles mosquitoes were collected using the standard pyrethrum spray catch and back-pack aspirator. Mosquito samples were sorted and morphologically identified to species level using international identification keys. Morphologically identified mosquito species were also confirmed using PCR. Genomic DNA was extracted from mosquitoes for molecular identification of blood meal source and parasite detection. The presence of Plasmodium species DNA in each mosquito sample was investigated using semi-nested PCR. Frequency of each blood meal source, Anopheles mosquito vector, and malaria parasite detected was calculated. Positivity rate of each fed female Anopheles mosquito was calculated for each species. RESULTS: A total of 2132 Anopheles mosquitoes were collected. 571 (26.8%) were males and 1561 (73.2%) were females classified based on their abdominal status into 1048 (67.1%) gravid, 274 (17.6%) fed, and 239 (15.3%) unfed females. Among the blood fed Anopheles mosquitoes, 263 (96.0%) were morphologically identified and confirmed using PCR to Anopheles arabiensis, 9 (3.3%) to Anopheles stephensi, and 2 (0.7%) to Anopheles rufipes. Of 274 blood-fed An. arabiensis, 68 (25.9%) fed on mixed blood meals from human and cattle, 8 (3.0%) fed on cattle and goat, and 13 (4.8%) fed on human, cattle and goat. For single blood meal sources, 70 (26.6%) fed on human, 95 (36.1%) fed on cattle, 8 (3.0%) fed on goat, and 1 (0.4%) fed on dog. While An. rufipes and An. stephensi fed on dog (2; 0.75%) and cattle (9; 3.3%), respectively. Plasmodium parasite detection in the blood meals showed that 25/274 (9.1%) An. arabiensis meals were positive for Plasmodium vivax and 19/274 (6.9%) An. arabiensis meals were positive for Plasmodium falciparum. The rate of positivity of An. arabiensis with any Plasmodium species was 16.7%. However, the positivity rate with P. falciparum only was 7.2%, while P. vivax was 9.5%. Both An. rufipes and An. stephensi were having positivity rates of 0.0% each. CONCLUSIONS: This study which was mainly on blood-fed Anopheles mosquitoes showed a diversity in the type of diet from human, cattle, and goat. Anopheles mosquitoes especially An. arabiensis in Sudan, are opportunistic blood feeders and can feed broadly on both human and cattle. The application of blood meal identification is not only important in malaria vector epidemiological surveillance but also is very useful in areas where arthropods exhibit zoophilic feeding behaviour for mammals.

Madurella mycetomatis grains within a eumycetoma lesion are clonal.

Eumycetoma is a neglected tropical infection of the subcutaneous tissue, characterized by tumor-like lesions and most commonly caused by the fungus Madurella mycetomatis. In the tissue, M. mycetomatis organizes itself in grains, and within a single lesion, thousands of grains can be present. The current hypothesis is that all these grains originate from a single causative agent, however, this hypothesis was never proven. Here, we used our recently developed MmySTR assay, a highly discriminative typing method, to determine the genotypes of multiple grains within a single lesion. Multiple grains from surgical lesions obtained from 11 patients were isolated and genotyped using the MmySTR panel. Within a single lesion, all tested grains shared the same genotype. Only in one single grain from one patient, a difference of one repeat unit in one MmySTR marker was noted relative to the other grains from that patient. We conclude that within these lesions the grains originate from a single clone and that the inherent unstable nature of the microsatellite markers may lead to small genotypic differences. LAY ABSTRACT: In lesions of the implantation mycosis mycetoma many Madurella mycetomatis grains are noted. It was unknown if grains arose after implantation of a single isolate or a mixture of genetically diverse isolates. By typing the mycetoma grains we showed that all grains within a single lesion were clonal and originated from a single isolate.

Assessment of genetic diversity of Plasmodium falciparum circumsporozoite protein in Sudan: the RTS,S leading malaria vaccine candidate.

BACKGROUND: The currently used malaria vaccine, RTS,S, is designed based on the Plasmodium falciparum circumsporozoite protein (PfCSP). The pfcsp gene, besides having different polymorphic patterns, can vary between P. falciparum isolates due to geographical origin and host immune response. Such aspects are essential when considering the deployment of the RTS,S vaccine in a certain region. Therefore, this study assessed the genetic diversity of P. falciparum in Sudan based on the pfcsp gene by investigating the diversity at the N-terminal, central repeat, and the C-terminal regions. METHODS: A cross-sectional molecular study was conducted; P. falciparum isolates were collected from different health centres in Khartoum State between January and December 2019. During the study period, a total of 261 febrile patients were recruited. Malaria diagnosis was made by expert microscopists using Giemsa-stained thick and thin blood films. DNA samples were examined by the semi-nested polymerase chain reaction (PCR). Single clonal infection of the confirmed P. falciparum cases, were used to amplify the pfcsp gene. The amplified amplicons of pfcsp have been sequenced using the Sanger dideoxy method. The obtained sequences of pfcsp nucleotide diversity parameters including the numbers of haplotypes (Hap), haplotypes diversity (Hapd), the average number of nucleotide differences between two sequences (p), and the numbers of segregating sites (S) were obtained. The haplotype networks were constructed using the online tcsBU software. Natural selection theory was also tested on pfcsp using Fuand Li's D, Fuand Li's F statistics, and Tajima's D test using DnaSP. RESULTS: In comparison with the different pfcsp reference strains, the Sudanese isolates showed high similarity with other African isolates. The results of the N-terminal region showed the presence of 2 different haplotypes with a Hapd of 0.425 ± 0.00727. The presence of the unique insertion of NNNGDNGREGKDEDKRDGNN was reported. The KLKQP motif was conserved in all the studied isolates. At the central repeat region, 11 haplotypes were seen with a Hapd of 0.779 ± 0.00097. The analysis of the genetic diversity in the C-terminal region showed the presence of 10 haplotypes with a Hapd of 0.457 ± 0.073. Several non-synonymous amino acids changes were also seen at the Th2R and the Th3R T-cell epitope regions including T317K, E317K, Q318E, K321N, I322K, T322K, R322K, K324Q, I327L, G352N, S354P, R355K, N356D, Q357E, and E361A. CONCLUSIONS: In this study, the results indicated a high conservation at the pfcsp gene. This may further contribute in understanding the genetic polymorphisms of P. falciparum prior to the deployment of the RTS,S vaccine in Sudan.

Parasitic contamination of fresh vegetables sold at central markets in Khartoum state, Sudan.

BACKGROUND: Fresh vegetables are considered as vital nutrients of a healthy diet as they supply the body with essential supplements. The consumption of raw vegetables is the main way for transmission of intestinal parasitic organisms. This study was aimed at detecting the parasitic contamination in fresh vegetables sold in two central open-aired markets in Khartoum state, Sudan. METHODS: In this prospective cross-sectional study, a total of 260 fresh vegetable samples and 50 water samples used to sprinkle vegetable(s) were collected from two central open-aired markets (namely; Elshaabi and Central markets) during November 2011 to May 2012. The samples were microscopically examined for detection of parasitic life forms using standardized parasitological techniques for protozoans and helminthes worms. RESULTS: Of the 260 fresh vegetable samples, 35 (13.5 %) were microscopically positive for intestinal parasites whereas 7/50 (14 %) of water samples used to sprinkle vegetable(s) were found positives. Remarkably, high level of contamination in fresh vegetable samples was recorded in lettuce (Lactuca sativa) 36.4 % (4/11) while cayenne pepper (Capsicum annuum) and cucumber (Cucumis sativus) were not contaminated. The identified protozoans and helminthes were Entamoeba histolytica/dispar, Entamoeba coli, Giardia lamblia, Ascaris lumbricoides, Strongyloides stercoralis, T. trichiura and hookworms. The most predominant parasite encountered was E. histolytica/dispar (42.9 %) whereas both T. trichiura and A. lumbricoides (2.9 %) were the least detected parasites. None of the fresh vegetables had single parasitic contamination. The highest percentages found in water samples used to sprinkle vegetable(s) was for Strongyloides larvae 60 % (3/5). It is worth-mentioned that the rate of contamination in Elshaabi market was higher compared with Central market. However, there was no significant correlation between the type of vegetables and existence of parasites in both markets and a high significant relationship was observed between the type of parasite and total prevalence in fresh vegetables (p = 0.000). CONCLUSION: The study has identified a moderate rate of fresh vegetables contaminated with protozoan and helminthes. Contaminated fresh vegetables in central markets of Khartoum state may play a significant role in transmission of intestinal parasitic infections to humans, and the water used by greengrocers to sprinkle vegetable(s) can be implicated in vegetable contamination.

Severe coinfection of dengue and malaria: A case report.

Key Clinical Message: In countries like Sudan, where several infectious diseases are prevalent, health care providers should not be satisfied with initial detection of a single pathogen and whenever it is feasible, they should investigate coinfections. Infections with high mortality or severe morbidity should be prioritized during the differential diagnosis particularly for diseases with similar clinical manifestations to reduce the death and disability rates. However, this requires substantial improvement in the diagnostic capacity. Abstract: Here we report a case of dengue and malaria coinfection from the southeast region of Sudan, bordering Ethiopia and Eritrea. A 25-year-old male from Sudan presented with symptoms of fever, chills, vomiting, and muscle and joint pain. Laboratory investigations confirmed a coinfection of dengue and malaria, which is assumingly not uncommon in areas heavily syndemic with several diseases but it is severely under-detected, underreported, and underestimated. The case has fully recovered after the supportive care for dengue and chemotherapy treatment for malaria. In such a case, it was important to monitor the patient's recovery and the treatment outcome through clinical indicators and laboratory parameters to update the treatment course whenever needed, according to response. The increasing burden and outbreaks of vector-borne diseases including dengue and malaria in Sudan, indicates the need for improving the implementation of the global vector control response that established by the World Health Organization. Additionally, the increasing prevalent of coinfections is urging substantial improvement in the diagnostic capacity in endemic countries.

Guillain-Barre syndrome associated with hepatitis E virus infection: A case report.

Key Clinical Message: Hepatitis E virus (HEV) infection can be manifested with several neurological syndromes including GBS. Therefore, healthcare providers should consider HEV in their differential diagnosis for patients with neurological disorders. Abstract: We report a case of Guillain-Barré syndrome associated with hepatitis E virus infection. The current case-report demonstrates diagnostic challenge to identify GBS case in a limited-resources country like Sudan. However, HEV infection should be highly suspected in patients with neurological manifestation with high liver enzymes.

The Rise of AI: How Artificial Intelligence is Revolutionizing Infectious Disease Control.

Artificial Intelligence (AI) has proven to be an effective tool in predicting, detecting, and mitigating the spread of infectious diseases, especially during the COVID-19 pandemic. The technology is increasingly playing a role in preventing future health crises by predicting outbreaks, identifying high-risk areas, and aiding in vaccine development. AI can track and trace infected individuals and identify potential hotspots, help reduce the spread of infectious diseases, and monitor patient symptoms, enabling healthcare professionals to provide effective treatment.

Rift Valley fever and malaria co-infection: A case report.

Key Clinical Message: We report a case of febrile illness that was presented with mild symptoms. However, laboratory investigation confirmed a malaria and Rift Valley fever co-infection. Healthcare providers in settings endemic with several infectious diseases should seek rolling out possibilities of other infections prior to starting treatment for achieving effective case management with less resources and better safety of patients. Abstract: Here we report a case of febrile illness that confirmed to be a co-infection of malaria and Rift Valley fever. The patient was initially diagnosed with malaria and started on treatment immediately. However, due to the lack of response to the treatment further laboratory investigations were pursued.

Cutaneous tuberculosis of the foot clinically mimicking mycetoma: A case report.

Key Clinical Message: In a resource-limited setting such as Sudan, where diagnostic and surveillance capacities are limited and several dermal diseases with similar clinical presentation are endemic, further precautious must taking into account. Abstract: Cutaneous tuberculosis (CTB) is a rare infection caused by Mycobacterium tuberculosis. Atypical clinical presentations of CTB may resemble other skin neglected diseases. For definitive diagnosis, we require a holistic diagnostic approach including clinical examination and deployment of laboratory investigations including microbial culture, histopathological, and molecular examinations of the proper samples per test. In this communication, we report a case of CTB that was initially misidentified clinically as mycetoma.

Sudden emergence and spread of cutaneous larva migrans in Sudan: A case series calls for urgent actions.

Cutaneous larva migrans (CLM) is a self-limiting dermatosis parasitic disease that is caused by zoonotic hookworm mainly infects cats, dogs, and accidentally humans. The disease affects hosts through the invasion and the migration of the hookworm larva into top layers of the skin. The disease occurs in tropical and subtropical regions; where people commonly are infected when sitting or barefoot walking on places contaminated with feces of infected cats or dogs. Due to self-limiting nature of the disease, the exact burden and prevalence are commonly underestimated. In this communication, we investigated all cases presented to the skin-diseases outpatient clinic' at the reference hospital of Tropical Diseases Medicine in Khartoum state between January 2019 and January 2021. This is the first-ever a case series report of cutaneous larva migrans in Sudan. We identified 15 cases of CLM presented with rash (100 %), redness of skin (67 %), and only adults were presented with larva crawling under their skins (27 %). Infection sites were (53 %) leg, (40 %) foot, only 7 % showed abdominal infection. The majority of patients were children or young adults, 47 % of them are ≤5 year-old, male to female ratio is 2.75:1. Duration of infection was one to three weeks and all patients fully recovered after treatment with albendazole. This urges for One Health interventions including deworming cats and dogs, improving water, sanitation, and hygiene, community-engagement and raising awareness in area with risk of infection.

The first Helicobacter pylori-induced Guillain-Barré syndrome in Sudan.

Key Clinical Message: This case report highlights the role of Helicobacter pylori infection in the development of GBS. Healthcare providers should consider H. pylori in their differential diagnosis for patients with neurological syndromes. Astract: We report the first known case of Guillain-Barré syndrome (GBS) associated with Helicobacter pylori infection in Sudan. This case highlights the role of H. pylori infection in the development of GBS. It also emphasizes the importance of wide screening of different endemic infections for patients with neurological syndromes for early detection and improves the case management in resource-limited settings like Sudan. Further research is needed to better understand the underlying mechanisms of H. pylori-inducing neurological disorders.

Recurrent abdominal wall mass in a hepatitis B-positive male: An unusual case of lumbar mycetoma.

Key Clinical Message: Atypical presentations of eumycetoma can pose a challenge in the diagnosis and treatment of the disease. Healthcare providers thorough in their differential diagnosis and investigations, even in the absence of classic symptoms, in order to improve early detection and the case management for such a neglected tropical disease. Abstract: In this communication, we present a case study of an unusual presentation of eumycetoma; a fungal infection that is considered a neglected tropical disease. The patient, a 28-year-old male from Sudan, presented with a recurrent mass in the abdominal wall. Despite two surgeries to remove the mass, it continued to recur. Unlike typical cases of eumycetoma, this patient did not exhibit common symptoms such as painless swelling, sinuses, or grain-containing discharge. The diagnosis was made incidentally after surgical excision of the mass. The abstract highlights the importance of recognizing uncommon presentations and maintaining a high suspicion for rare diagnoses, even in the absence of classic symptoms. Further research is needed to better understand atypical presentations of eumycetoma and improve early detection.

Unusual manifestation of cystic mycetoma lesions: A case report.

Key Clinical Message: This case presents an atypical cystic presentation of mycetoma without sinuses or discharge. Awareness of these variations is crucial for accurate diagnosis and timely intervention, highlighting the need for healthcare professionals to consider diverse manifestations of mycetoma. Abstract: Mycetoma is a chronic and debilitating infectious disease characterized by localized swellings and granulomatous lesions. It primarily affects individuals in tropical and subtropical regions and is caused by certain fungi or bacteria. While mycetoma typically presents with sinuses and discharge, this case report presents a unique cystic presentation without these features. The patient, a 12-year-old female from Sudan, presented with a painless swelling on the dorsum of her right foot. Physical examination revealed a round, non-tender, and fluctuant mass. Histopathological examination confirmed actinomycetoma caused by Streptomyces somaliensis. The patient was successfully treated with a combination of antibiotherapy. This atypical presentation underscores the need for healthcare professionals to consider uncommon variations of mycetoma for accurate diagnosis and management.

Specific and sensitive loop-mediated isothermal amplification (LAMP) method for Madurella strains, eumycetoma filamentous fungi causative agent.

BACKGROUND: Filamentous fungi of the genus Madurella are the primary causative agents of mycetoma, a disease observed in tropical and subtropical regions. Since early diagnostics based on a morphological approach are difficult and have many shortcomings, a molecular diagnostic method suitable for rural settings is required. In this study, we developed the loop-mediated isothermal amplification (LAMP) method to present a foundational technique of the diagnosis of Madurella spp. (M. mycetomatis, M. pseudomycetomatis, M. tropicana, and M. fahalii), the common causative organisms of eumycetoma. PRINCIPAL FINDINGS: We successfully designed a primer pair targeting the rDNAs of three Madurella spp. excluding M. fahalii, and detected up to 100 fg of genomic DNA extracted from isolates of M. mycetomatis and 1 pg of M. pseudomycetomatis and M. tropicana, within one hour. Second, a primer pair specific to M. mycetomatis, the most common causative species, or M. fahalii, a drug-resistant species, was constructed, and the detection limit of both primer pairs was 1 pg. The designed primers accurately distinguished 16 strains of the genus Madurella from various fungal species known to cause mycetomas. CONCLUSION: In summary, we established the first model of a LAMP detection method that rapidly and sensitively detects and identifies Madurella isolates for clinical diagnostics. Moreover, the combined designed primer sets could identify mycetoma-causing strains simultaneously.

The First Case of Fusarium falciforme Eumycetoma in Sudan and an Extensive Literature Review about Treatment Worldwide.

Eumycetoma is an infectious disease caused by various fungal pathogens. The disease is characterised by black and pale-yellowish grain discharge. In this communication, we report a case of eumycetoma with a pale grain foot-eumycetoma caused by Fusarium falciforme. The patient presented at the outpatient clinic of the Mycetoma Research Centre in Sudan. The causative agent was initially misidentified as Aspergillus nidulans based on its seemingly similar histopathological appearance. However, sequencing the internally transcribed spacer region of the extracted grain confirmed infection with Fusarium falciforme. Although the patient received Itraconazole and underwent surgical excision, the disease was recurrent. To our knowledge, this is the first report on Fusarium falciforme causing eumycetoma in Sudan, indicating the expansion of the geographical distribution of this pathogen. This calls for raising the awareness of healthcare providers and improving the diagnostic and surveillance systems in at-risk areas to improve the case management and reduce the threat of further spread. Considering the potential impacts of F. falciforme infection including threatening the global health, food security, and ecosystem balance, as well as loss of biodiversity and negative socioeconomic changes in endemic countries, we recommend the implementation of an integrated transdisciplinary One Health strategy for the prevention and control of emerging infectious diseases including F. falciforme.