Wendy W. J. van de Sande

Mycetoma caused by Madurella mycetomatis: a neglected infectious burden

Tropical eumycetoma is frequently caused by the fungus Madurella mycetomatis. The disease is characterised by extensive subcutaneous masses, usually with sinuses draining pus, blood, and fungal grains. The disease affects individuals of all ages, although disability is most severe in adults who work outdoors. Compared with major diseases such as tuberculosis, malaria, and HIV, disease from M mycetomatis is underestimated but socioeconomically important. Many scientific case reports on mycetoma exist, but fundamental research was lacking until recently. We present a review on developments in the clinical, epidemiological, and diagnostic management of M mycetomatis eumycetoma. We describe newly developed molecular diagnostic and gene typing procedures, and their application for management of patients and environmental research. Fungal susceptibility tests have been developed as well as a mouse model of infection. These advances should greatly further our understanding of the molecular basis of eumycetoma.

Global Burden of Human Mycetoma: A Systematic Review and Meta-analysis

Mycetoma is a chronic infectious disease of the subcutaneous tissue with a high morbidity. This disease has been reported from countries between 30°N and 15°S since 1840 but the exact burden of disease is not known. It is currently unknown what the incidence, prevalence and the number of reported cases per year per country is. In order to estimate what the global burden of mycetoma is, a meta-analysis was performed. In total 50 studies were included, which resulted in a total of 8763 mycetoma cases. Most cases were found in men between 11 and 40 years of age. The foot was most commonly affected. Most cases were reported from Mexico, Sudan and India. Madurella mycetomatis was the most prevalent causative agent world-wide, followed by Actinomadura madurae, Streptomyces somaliensis, Actinomadura pelletieri, Nocardia brasiliensis and Nocardia asteroides. Although this study represents a first indication of the global burden on mycetoma, the actual burden is probably much higher. In this study only cases reported to literature could be used and most of these cases were found by searching archives from a single hospital in a single city of that country. By erecting (inter)national surveillance programs a more accurate estimation of the global burden on mycetoma can be obtained.

Aspergillus fumigatus conidial melanin modulates host cytokine response.

Melanin biopigments have been linked to fungal virulence. Aspergillus fumigatus conidia are melanised and are weakly immunogenic. We show that melanin pigments on the surface of resting Aspergillus fumigatus conidia may serve to mask pathogen-associated molecular patterns (PAMPs)-induced cytokine response. The albino conidia induced significantly more proinflammatory cytokines in human peripheral blood mononuclear cells (PBMC), as compared to melanised wild-type conidia. Blocking dectin-1 receptor, Toll-like receptor 4 or mannose receptor decreased cytokine production induced by the albino but not by the wild type conidia. Moreover, albino conidia stimulated less potently, cytokine production in PBMC isolated from an individual with defective dectin-1, compared to the stimulation of cells isolated from healthy donors. These results suggest that β-glucans, but also other stimulatory PAMPs like mannan derivatives, are exposed on conidial surface in the absence of melanin. Melanin may play a modulatory role by impeding the capability of host immune cells to respond to specific ligands on A. fumigatus.

Management of mycetoma: major challenge in tropical mycoses with limited international recognition

Purpose of review The present review highlights an orphan infectious disease in alarming need of international recognition. While money is being invested to develop new broad-spectrum antimicrobial drugs to treat infection in general, improvement in the management of complicated infections such as mycetoma receives little support. Recent findings Many case presentations describe single-center experience in the management of mycetoma. Unfortunately, randomized and blinded clinical studies into the efficacy of antimicrobial treatment are desperately lacking. Response to medical treatment is usually better in actinomycetoma than eumycetoma. Eumycetoma is difficult to treat using current therapies. Surgery in combination with azole treatment is the recommended regimen for small eumycetoma lesions in the extremities. Bone involvement complicates clinical management, leaving surgical amputation as the only treatment option. Although clinical management has not received major attention recently, laboratory technology has improved in areas of molecular diagnosis and epidemiology. Summary Management of mycetoma and laboratory diagnosis of its etiological agents need to be improved and better implemented in endemic regions. Optimized therapeutic approaches and more detailed epidemiological data are urgently needed. It is vital to initiate multicenter collaborations on national and international levels to develop consensus clinical score sheets and state-of-the-art treatment regimens for mycetoma patients.

Antifungal Susceptibility and Phylogeny of Opportunistic Members of the Order Mucorales

ABSTRACT The in vitro susceptibilities of 66 molecularly identified strains of the Mucorales to eight antifungals (amphotericin B, terbinafine, itraconazole, posaconazole, voriconazole, caspofungin, micafungin, and 5-fluorocytosine) were tested. Molecular phylogeny was reconstructed based on the nuclear ribosomal large subunit to reveal taxon-specific susceptibility profiles. The impressive phylogenetic diversity of the Mucorales was reflected in susceptibilities differing at family, genus, and species levels. Amphotericin B was the most active drug, though somewhat less against Rhizopus and Cunninghamella species. Posaconazole was the second most effective antifungal agent but showed reduced activity in Mucor and Cunninghamella strains, while voriconazole lacked in vitro activity for most strains. Genera attributed to the Mucoraceae exhibited a wide range of MICs for posaconazole, itraconazole, and terbinafine and included resistant strains. Cunninghamella also comprised strains resistant to all azoles tested but was fully susceptible to terbinafine. In contrast, the Lichtheimiaceae completely lacked strains with reduced susceptibility for these antifungals. Syncephalastrum species exhibited susceptibility profiles similar to those of the Lichtheimiaceae. Mucor species were more resistant to azoles than Rhizopus species. Species-specific responses were obtained for terbinafine where only Rhizopus arrhizus and Mucor circinelloides were resistant. Complete or vast resistance was observed for 5-fluorocytosine, caspofungin, and micafungin. Intraspecific variability of in vitro susceptibility was found in all genera tested but was especially high in Mucor and Rhizopus for azoles and terbinafine. Accurate molecular identification of etiologic agents is compulsory to predict therapy outcome. For species of critical genera such as Mucor and Rhizopus, exhibiting high intraspecific variation, susceptibility testing before the onset of therapy is recommended.

Validation of a New Aspergillus Real-Time PCR Assay for Direct Detection of Aspergillus and Azole Resistance of Aspergillus fumigatus on Bronchoalveolar Lavage Fluid

ABSTRACT Azole resistance in Aspergillus fumigatus is increasingly reported. Here, we describe the validation of the AsperGenius, a new multiplex real-time PCR assay consisting of two multiplex real-time PCRs, one that identifies the clinically relevant Aspergillus species, and one that detects the TR34, L98H, T289A, and Y121F mutations in CYP51A and differentiates susceptible from resistant A. fumigatus strains. The diagnostic performance of the AsperGenius assay was tested on 37 bronchoalveolar lavage (BAL) fluid samples from hematology patients and 40 BAL fluid samples from intensive care unit (ICU) patients using a BAL fluid galactomannan level of ≥1.0 or positive culture as the gold standard for detecting the presence of Aspergillus. In the hematology and ICU groups combined, there were 22 BAL fluid samples from patients with invasive aspergillosis (IA) (2 proven, 9 probable, and 11 nonclassifiable). Nineteen of the 22 BAL fluid samples were positive, according to the gold standard. The optimal cycle threshold value for the presence of Aspergillus was <36. Sixteen of the 19 BAL fluid samples had a positive PCR (2 Aspergillus species and 14 A. fumigatus samples). This resulted in a sensitivity, specificity, and positive and negative predictive values of 88.9%, 89.3%, 72.7%, and 96.2%, respectively, for the hematology group and 80.0%, 93.3%, 80.0%, and 93.3%, respectively, in the ICU group. The CYP51A real-time PCR confirmed 12 wild-type and 2 resistant strains (1 TR34-L98H and 1 TR46-Y121F-T289A mutant). Voriconazole therapy failed for both patients. The AsperGenius multiplex real-time PCR assay allows for sensitive and fast detection of Aspergillus species directly from BAL fluid samples. More importantly, this assay detects and differentiates wild-type from resistant strains, even if BAL fluid cultures remain negative.

Testing of the In Vitro Susceptibilities of Madurella mycetomatis to Six Antifungal Agents by Using the Sensititre System in Comparison with a Viability-Based 2,3-Bis(2-Methoxy-4-Nitro-5-Sulfophenyl)-5- [(Phenylamino)Carbonyl]-2H-Tetrazolium Hydroxide (XTT) Assay and a Modified NCCLS Method

ABSTRACT The in vitro susceptibilities of 36 clinical isolates of Madurella mycetomatis, the prime agent of eumycetoma in Africa, to ketoconazole, itraconazole, fluconazole, voriconazole, amphotericin B, and flucytosine were determined by the Sensititre YeastOne system. This system appeared to be a rapid and easy test, and by use of hyphal suspensions it generated results comparable to those of a modified NCCLS method. After 10 days of incubation, the antifungal activities of ketoconazole (MIC at which 90% of isolates were inhibited [MIC90], 0.125 μg/ml), itraconazole (MIC90, 0.064 μg/ml), and voriconazole (MIC90, 0.125 μg/ml) appeared superior to those of fluconazole (MIC90, 128 μg/ml) and amphotericin B (MIC90, 1 μg/ml), with MICs in the clinically relevant range. All isolates were resistant to flucytosine (all MICs above 64 μg/ml). Based on the relatively broad range of MICs obtained for the antifungal agents, routine testing of M. mycetomatis isolates for susceptibility to antifungal agents seems to be relevant to adequate therapeutic management.

In Vitro Susceptibilities of Madurella mycetomatis to Itraconazole and Amphotericin B Assessed by a Modified NCCLS Method and a Viability-Based 2,3-Bis(2-Methoxy-4-Nitro-5- Sulfophenyl)-5-[(Phenylamino)Carbonyl]-2H- Tetrazolium Hydroxide (XTT) Assay

ABSTRACT Susceptibilities of Madurella mycetomatis against amphotericin B and itraconazole in vitro were determined by protocols based on NCCLS guidelines (visual reading) and a 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) assay for fungal viability. The XTT assay was reproducible and sensitive for both antifungals. Itraconazole (MIC at which 50% of the isolates tested are inhibited [MIC50]) of 0.06 to 0.13 mg/liter) was superior to amphotericin B (MIC50 of 0.5 to 1.0 mg/liter).

Diagnosis of active tuberculosis by e-nose analysis of exhaled air

Tuberculosis (TB), a highly infectious airborne disease, remains a major global health problem. Many of the new diagnostic techniques are not suited for operation in the highly-endemic low-income countries. A sensitive, fast, easy-to-operate and low-cost method is urgently needed. We performed a Proof of Principle Study (30 participants) and a Validation Study (194 participants) to estimate the diagnostic accuracy of a sophisticated electronic nose (DiagNose, C-it BV) using exhaled air to detect tuberculosis. The DiagNose uses a measurement method that enables transfer of calibration models between devices thus eliminating the most common pitfall for large scale implementation of electronic noses in general. DiagNose measurements were validated using traditional sputum smear microscopy and culture on Löwenstein-Jensen media. We found a sensitivity of 95.9% and specificity of 98.5% for the pilot study. In the validation study we found a sensitivity of 93.5% and a specificity of 85.3% discriminating healthy controls from TB patients, and a sensitivity of 76.5% and specificity of 87.2% when identifying TB patient within the entire test-population (best-case numbers). The portability and fast time-to-result of the DiagNose enables a proactive screening search for new TB cases in rural areas, without the need for highly-skilled operators or a hospital center infrastructure.

Mycetoma: a unique neglected tropical disease

Mycetoma can be caused by bacteria (actinomycetoma) or fungi (eumycetoma) and typically affects poor communities in remote areas. It is an infection of subcutaneous tissues resulting in mass and sinus formation and a discharge that contains grains. The lesion is usually on the foot but all parts of the body can be affected. The causative microorganisms probably enter the body by a thorn prick or other lesions of the skin. Mycetoma has a worldwide distribution but is restricted to specific climate zones. Microbiological diagnosis and characterisation of the exact organism causing mycetoma is difficult; no reliable serological test exists but molecular techniques to identify relevant antigens have shown promise. Actinomycetoma is treated with courses of antibiotics, which usually include co-trimoxazole and amikacin. Eumycetoma has no acceptable treatment at present; antifungals such as ketoconazole and itraconazole have been used but are unable to eradicate the fungus, need to be given for long periods, and are expensive. Amputations and recurrences in patients with eumycetoma are common.