Molecular and biological studies on Madurella mycetomatis infection in human and mice
Madurella mycotomatis infecties in mensen en muizen, moleculaire en biologische studies
Madurella mycetomatis is the most common cause of eumycetoma in tropical and sub tropical countries. The mycetoma infection is a serious mycological problem affecting a large number of active members of the society in endemic areas. The infection is difficult to treat and has an important social and economical impact on affected patients. M. mycetomatis is a fungus showing poor morphological differentiation, and identification of the species is always difficult. The early diagnosis is one of the key elements mandatory to successful treatment of mycetoma. This, however, is not easy due to the many limitations of the currently used diagnostic techniques such as direct examination of grains, culture and histopathology. In an attempt to improve the diagnosis, we developed a PCR-RFLP procedure for the detection and identification of the fungus in cultures and clinical specimens. The M. mycetomatis ribosomal gene complex was amplified and sequenced using fungal universal primers. Two sets of species-specific PCR primers were designed. Based on ITS sequences and RFLP patterns, clinical isolates from Sudan were found to belong to a single species. In chapter 3, complicated eumycetoma cases were reported, in which the etiological agents were identified using diagnostic PCR and DNA sequencing. The novel PCR primers were also used for the study of the environmental occurrence of M. mycetomatis in endemic regions. Direct cuture-based isolation of M. mycetomatis from the patient's surrounding environment failed, but DNA was detected in 25% of the environmental specimens (chapter 4). The phylogenetic relationship of M. mycetomatis to morphologically similar fungal species was also studied, and its taxonomic position in the fungal kingdom was redefined. Our results suggested that M. mycetomatis belongs to the ascomycete order Sordariales, while the other proposed member of the same genus "Madurella grisea" likely to be a member of the order Pleosporales (chapter 5). In order to establish the genetic relatedness of M. mycetomatis clinical isolates, a collection of isolates from two endemic foci in Africa were analyzed using large-scale RAPD typing. These analyses showed a complete lack of DNA fingerprint variation between the various isolates. These results suggest that there is little genetic variation among clinically relevant M. mycetomatis strains from the regions involved (chapter 6). A reproducible infection model for M. mycetomatis in BALB/c mice was developed. Different routes of inoculation, adjuvant types, host immune status, and gender of mice were evaluated. The infection was found to be inoculum-dependent with increased infection rates for larger inocula. Adjuvants were needed for the induction of the infection (chapter 7). The antifungal susceptibility of M. mycetomatis to amphotericin B and itraconazole was studied using two new protocols, one adapted from the NCCLS approved guidelines M38-A, the other assay was developed to quantify the antifungal activity. Most of the M. mycetomatis clinical isolates were highly susceptible to itraconazole, but not to amphotericin B (chapter 8). In conclusion, sequence data from the M. mycetomatis rONA were used to develop a novel diagnostic PCR-RFLP procedure for the identification of M. mycetomatis in cultures, clinical specimens, the environment and for solving diagnostic problems with unexpected, complex patient cases. In addition, molecular biological techniques enabled us to study the ecology, taxonomy and phylogeny of M. mycetomatis. An infection model in mice was developed and is now ready to be used for the study of the pathology, immunology and the in vivo susceptibility to common antifungal agents of M. mycetomatis. A sound basis for future research into (early) serodiagnosis, host susceptibility towards M. mycetomatis infection and in vivo therapeutic studies has been laid.
|Keywords||Madurella mycetomatis, diagnsitics, human, mice, infection diseases, epidemiology, fungal viability|
|Promotor||H.A. Verbrugh (Henri) , A.H. Fahal (Ahmed) , A.F. van Belkum (Alex)|
|Publisher||Erasmus University Rotterdam|
Ahmed, A.A.O. (2003, December 17). Molecular and biological studies on Madurella mycetomatis infection in human and mice. Erasmus University Rotterdam. Retrieved from http://hdl.handle.net/1765/51365