Gene expression of Paracoccidioides virulence factors after interaction with macrophages and fibroblasts.

BACKGROUND: Paracoccidioidomycosis (PCM) is a systemic mycosis with high prevalence in Latin America that is caused by thermodimorphic fungal species of the Paracoccidioides genus. OBJECTIVES: In this study, we used quantitative polymerase chain reaction (qPCR) to investigate the expression of genes related to the virulence of Paracoccidioides brasiliensis (Pb18) and P. lutzii (Pb01) strains in their mycelial (M) and yeast (Y) forms after contact with alveolar macrophages (AMJ2-C11 cell line) and fibroblasts (MRC-5 cell line). METHODS: The selected genes were those coding for 43 kDa glycoprotein (gp43), enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 14-3-3 protein (30 kDa), phospholipase, and aspartyl protease. FINDINGS: In the Pb18 M form, the aspartyl protease gene showed the highest expression among all genes tested, both before and after infection of host cells. In the Pb18 Y form after macrophage infection, the 14-3-3 gene showed the highest expression among all genes tested, followed by the phospholipase and gp43 genes, and their expression was 50-fold, 10-fold, and 6-fold higher, respectively, than that in the M form. After fibroblast infection with the Pb18 Y form, the 14-3-3 gene showed the highest expression, followed by the phospholipase and aspartyl protease genes, and their expression was 25-fold, 10-fold, and 10-fold higher, respectively, than that in the M form. Enolase and aspartyl protease genes were expressed upon infection of both cell lines. After macrophage infection with the Pb01 Y form, the 14-3-3 gene showed the highest expression, followed by the phospholipase and aspartyl protease genes, and their expression was 18-fold, 12.5-fold, and 6-fold higher, respectively, than that in the M form. MAIN CONCLUSIONS: In conclusion, the data show that the expression of the genes analysed may be upregulated upon fungus-host interaction. Therefore, these genes may be involved in the pathogenesis of paracoccidioidomycosis.

COVID-19: The question of genetic diversity and therapeutic intervention approaches.

Coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2), is the largest pandemic in modern history with very high infection rates and considerable mortality. The disease, which emerged in China's Wuhan province, had its first reported case on December 29, 2019, and spread rapidly worldwide. On March 11, 2020, the World Health Organization (WHO) declared the COVID-19 outbreak a pandemic and global health emergency. Since the outbreak, efforts to develop COVID-19 vaccines, engineer new drugs, and evaluate existing ones for drug repurposing have been intensively undertaken to find ways to control this pandemic. COVID-19 therapeutic strategies aim to impair molecular pathways involved in the virus entrance and replication or interfere in the patients' overreaction and immunopathology. Moreover, nanotechnology could be an approach to boost the activity of new drugs. Several COVID-19 vaccine candidates have received emergency-use or full authorization in one or more countries, and others are being developed and tested. This review assesses the different strategies currently proposed to control COVID-19 and the issues or limitations imposed on some approaches by the human and viral genetic variability.

Gentamicin encapsulated within a biopolymer for the treatment of Staphylococcus aureus and Escherichia coli infected skin ulcers.

Skin wound infection requires carefully long-term treatment with an immense financial burden to healthcare systems worldwide. Various strategies such as drug delivery systems using polymer matrix from natural source have been used to enhance wound healing. Natural rubber latex (NRL) from Hevea brasiliensis has shown angiogenic and tissue repair properties. Gentamicin sulfate (GS) is a broad-spectrum antibiotic which inhibits the growth of a wide variety of microorganisms and, because of this, it has also been applied topically for treatment of local infections. The aim of this study was to develop a GS release system using NRL as matrix for Staphylococcus aureus and Escherichia coli infected skin ulcers treatment, without changing drug antibiotic properties. The matrix did not change the GS antimicrobial activity against S. aureus and E. coli strains. Moreover, the NRL-GS biomembrane did not exhibit hemolytic activity, being non-toxic to red blood cells. The eluates of NRL-GS biomembranes and GS solutions did not significantly reduce the survival of Caenorhabditis elegans worms for 24 h at any of the tested concentrations. Thus, these results emphasize that the NRL-GS biomembrane proved to be a promising biomaterial for future studies on the development of dressings for topical uses, inexpensive and practicable, keeping drug antibiotic properties against pathogens and to reduce the side effects.

Natural rubber latex membranes incorporated with three different types of propolis: Physical-chemistry and antimicrobial behaviours.

Natural Rubber Latex (NRL) is a biocompatible material with demonstrated capacity to induce vascularisation and tissue regeneration. Propolis is a complex resinous product prepared by Apis mellifera with the aim of protecting beehives against infectious microorganisms. It is flora-dependent and its antimicrobial activity can vary according to its geographical origin. This study compares the incorporation of three different types of propolis into an NRL membrane aiming at optimal controlled release of propolis potential antimicrobial compounds towards Candida albicans whilst keeping NRL mechanical characteristics desirable for wound healing bandage purposes. The propolis samples were classified as red, green and poplar propolis according to their chemical composition determined by ultra-high performance liquid chromatography coupled in series with both UV spectrophotometry and high-resolution mass spectrometry. The Minimum Inhibitory Concentrations (MIC) towards C. albicans were determined before their incorporation into NRL membranes. The release of NRL-propolis components in Simulated Body Fluid (SBF) was monitored by UV-Vis spectroscopy. The antimicrobial activity and the effects of the materials released on mouse fibroblasts were assessed. FTIR analyses were carried out in order to verify the formation of new chemical bonds that might prevent the release of propolis components from the NRL membrane. The mechanical characteristics of the NRL membranes remained adequate after the incorporation of the three types of propolis investigated whilst allowing the release of the red, and poplar propolis most active compounds against C. albicans. At 30 and 50% the released materials (eluates) from the NRL membranes incorporated with red and poplar propolis types were not toxic to fibroblast cells. These results suggest that red and poplar propolis can be incorporated into NRL membranes for the preparation of wound healing dressing.

Optimization of Antimicrobial Photodynamic Therapy in Biofilms by Inhibiting Efflux Pump.

BACKGROUND: One of the main mechanisms of microbial resistance is given by efflux pumps, which reduce the effectiveness of antimicrobials by decreasing their intracellular concentration. OBJECTIVE AND METHODS: Considering that efflux pump inhibitors are promising adjuvant molecules for antibiotics in infections, in this study, using XTT test and colony forming unit (CFU) counting, we evaluated the association between the pump inhibitor verapamil (VP) and the antimicrobial photodynamic therapy (aPDT) mediated by methylene blue (MB) in biofilms of Escherichia coli and Staphylococcus aureus to optimize the bacterial reduction. RESULTS: By applying 44 J/cm2, 215 µg/mL of VP, and 200 µg/mL of MB, we obtained 80% of metabolism reduction and 3.4 log10 CFU/mL decrease for E. coli. Biofilm of S. aureus presented 80% of metabolism reduction and 3.65 log10 CFU/mL decrease when 22 J/cm2, 312 µg/mL of VP, and 200 µg/mL of MB was used. Applying 200 µg/mL of MB, the E. coli biofilm required a higher dose of light, while the S. aureus biofilm required a higher concentration of VP to obtain the same reduction. CONCLUSIONS: The VP optimized the efficiency of aPDT and showed no toxicity when used alone in both strains, proving that inhibiting efflux pumps in combination with aPDT has great potential for clinical application.

Selective photoinactivation of Histoplasma capsulatum by water-soluble derivatives chalcones.

Histoplasmosis is a respiratory and systemic disease caused by the dimorphic fungus Histoplasma capsulatum. The clinical features may vary from asymptomatic infections to disseminated severe form depending of patient immunity. The treatment of histoplasmosis can be performed with itraconazole, fluconazole, and in the disseminated forms is used amphotericin B. However, the critical side effects of amphotericin B, the cases of itraconazole therapy failure and the appearance of fluconozole-resistant strains makes necessary the search of new strategies to treat this disease. Antimicrobial photodynamic therapy (aPDT) seems to be a potential candidate once have been show efficacy to inhibit others dimorphic fungi. Although the photosensitizer (PS) chalcone aggregates in biological medium, it has antifungal activity and show a high quantum yield of ROS formation. So, the aim of this study was to obtain the experimental parameters to achieve an acceptable selective chalcone water-soluble derivatives photoinactivation of H. capsulatum comparing with fibroblastic and keratinocytes cells which are the constituents of some potential host tissues. Yeast and cells were incubated with the same chalchones concentrations and short incubation time followed by irradiation with equal dose of light. The best conditions to kill H. capsulatum selectively were very low photosensitizers concentration (1.95µgmL-1) incubated by 15min and irradiated with LED 450nm with 24Jcm-2. Key words: chalcone, Histoplasma capsulatum, aPDT, selectivity.

A cell wall protein-based vaccine candidate induce protective immune response against Sporothrix schenckii infection.

Sporotrichosis is a subcutaneous mycosis caused by several closely related thermo-dimorphic fungi of the Sporothrix schenckii species complex, affecting humans and other mammals. In the last few years, new strategies have been proposed for controlling sporotrichosis owning to concerns about its growing incidence in humans, cats, and dogs in Brazil, as well as the toxicity and limited efficacy of conventional antifungal drugs. In this study, we assessed the immunogenicity and protective properties of two aluminum hydroxide (AH)-adsorbed S. schenckii cell wall protein (ssCWP)-based vaccine formulations in a mouse model of systemic S. schenckii infection. Fractioning by SDS-PAGE revealed nine protein bands, two of which were functionally characterized: a 44kDa peptide hydrolase and a 47kDa enolase, which was predicted to be an adhesin. Sera from immunized mice recognized the 47kDa enolase and another unidentified 71kDa protein, whereas serum from S. schenckii-infected mice recognized both these proteins plus another unidentified 9.4kDa protein. Furthermore, opsonization with the anti-ssCWP sera led to markedly increased phagocytosis and was able to strongly inhibit the fungus' adhesion to fibroblasts. Immunization with the higher-dose AH-adjuvanted formulation led to increased ex vivo release of IL-12, IFN-γ, IL-4, and IL-17, whereas only IL-12 and IFN-γ were induced by the higher-dose non-adjuvanted formulation. Lastly, passive transference of the higher-dose AH-adjuvanted formulation's anti-ssCWP serum was able to afford in vivo protection in a subsequent challenge with S. schenckii, becoming a viable vaccine candidate for further testing.

Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells.

The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis.

Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells

The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis.

Serological diagnosis of paracoccidioidomycosis: high rate of inter-laboratorial variability among medical mycology reference centers.

BACKGROUND: Serological tests have long been established as rapid, simple and inexpensive tools for the diagnosis and follow-up of PCM. However, different protocols and antigen preparations are used and the few attempts to standardize the routine serological methods have not succeeded. METHODOLOGY/PRINCIPAL FINDINGS: We compared the performance of six Brazilian reference centers for serological diagnosis of PCM. Each center provided 30 sera of PCM patients, with positive high, intermediate and low titers, which were defined as the "reference" titers. Each center then applied its own antigen preparation and serological routine test, either semiquantitative double immunodifusion or counterimmmunoelectrophoresis, in the 150 sera from the other five centers blindly as regard to the "reference" titers. Titers were transformed into scores: 0 (negative), 1 (healing titers), 2 (active disease, low titers) and 3 (active disease, high titers) according to each center's criteria. Major discordances were considered between scores indicating active disease and scores indicating negative or healing titers; such discordance when associated with proper clinical and other laboratorial data, may correspond to different approaches to the patient's treatment. Surprisingly, all centers exhibited a high rate of "major" discordances with a mean of 31 (20%) discordant scores. Alternatively, when the scores given by one center to their own sera were compared with the scores given to their sera by the remaining five other centers, a high rate of major discordances was also found, with a mean number of 14.8 sera in 30 presenting a discordance with at least one other center. The data also suggest that centers that used CIE and pool of isolates for antigen preparation performed better. CONCLUSION: There are inconsistencies among the laboratories that are strong enough to result in conflicting information regarding the patients' treatment. Renewed efforts should be promoted to improve standardization of the serological diagnosis of PCM.

Anti dermatophytic therapy: prospects for the discovery of new drugs from natural products

Millions of people and animals suffer from superficial infections caused by a group of highly specialized filamentous fungi, the dermatophytes, which only infect keratinized structures. With the appearance of AIDS, the incidence of dermatophytosis has increased. Current drug therapy used for these infections is often toxic, long-term, and expensive and has limited effectiveness; therefore, the discovery of new anti dermatophytic compounds is a necessity. Natural products have been the most productive source for new drug development. This paper provides a brief review of the current literature regarding the presence of dermatophytes in immunocompromised patients, drug resistance to conventional treatments and new anti dermatophytic treatments.

Paracoccidoides brasiliensis 30 kDa adhesin: identification as a 14-3-3 protein, cloning and subcellular localization in infection models.

Paracoccidoides brasiliensis adhesion to lung epithelial cells is considered an essential event for the establishment of infection and different proteins participate in this process. One of these proteins is a 30 kDa adhesin, pI 4.9 that was described as a laminin ligand in previous studies, and it was more highly expressed in more virulent P. brasiliensis isolates. This protein may contribute to the virulence of this important fungal pathogen. Using Edman degradation and mass spectrometry analysis, this 30 kDa adhesin was identified as a 14-3-3 protein. These proteins are a conserved group of small acidic proteins involved in a variety of processes in eukaryotic organisms. However, the exact function of these proteins in some processes remains unknown. Thus, the goal of the present study was to characterize the role of this protein during the interaction between the fungus and its host. To achieve this goal, we cloned, expressed the 14-3-3 protein in a heterologous system and determined its subcellular localization in in vitro and in vivo infection models. Immunocytochemical analysis revealed the ubiquitous distribution of this protein in the yeast form of P. brasiliensis, with some concentration in the cytoplasm. Additionally, this 14-3-3 protein was also present in P. brasiliensis cells at the sites of infection in C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells for 72 h (acute infections) and 30 days (chronic infection). An apparent increase in the levels of the 14-3-3 protein in the cell wall of the fungus was also noted during the interaction between P. brasiliensis and A549 cells, suggesting that this protein may be involved in host-parasite interactions, since inhibition assays with the protein and this antibody decreased P. brasiliensis adhesion to A549 epithelial cells. Our data may lead to a better understanding of P. brasiliensis interactions with host tissues and paracoccidioidomycosis pathogenesis.

Anti dermatophytic therapy--prospects for the discovery of new drugs from natural products.

Millions of people and animals suffer from superficial infections caused by a group of highly specialized filamentous fungi, the dermatophytes, which only infect keratinized structures. With the appearance of AIDS, the incidence of dermatophytosis has increased. Current drug therapy used for these infections is often toxic, long-term, and expensive and has limited effectiveness; therefore, the discovery of new anti dermatophytic compounds is a necessity. Natural products have been the most productive source for new drug development. This paper provides a brief review of the current literature regarding the presence of dermatophytes in immunocompromised patients, drug resistance to conventional treatments and new anti dermatophytic treatments.

Anticandidal efficacy of cinnamon oil against planktonic and biofilm cultures of Candida parapsilosis and Candida orthopsilosis.

Candida parapsilosis is yeast capable of forming biofilms on medical devices. Novel approaches for the prevention and eradication of the biofilms are desired. This study investigated the anticandidal activity of sixteen essential oils on planktonic and biofilm cultures of C. parapsilosis complex. We used molecular tools, enumeration of colony-forming units, the colourimetric MTT assay, scanning electron microscopy (SEM) and a chequerboard assay coupled with software analyses to evaluate the growth kinetics, architecture, inhibition and reduction in biofilms formed from environmental isolates of the Candida parapsilosis complex; further, we also evaluated whether essential oils would interact synergistically with amphotericin B to increase their anticandidal activities. Of the environmental C. parapsilosis isolates examined, C. parapsilosis and C. orthopsilosis were identified. Biofilm growth on polystyrene substrates peaked within 48 h, after which growth remained relatively stable up to 72 h, when it began to decline. Details of the architectural analysis assessed by SEM showed that C. parapsilosis complex formed less complex biofilms compared with C. albicans biofilms. The most active essential oil was cinnamon oil (CO), which showed anticandidal activity against C. orthopsilosis and C. parapsilosis in both suspension (minimum inhibitory concentration-MIC-250 and 500 µg/ml) and biofilm (minimum biofilm reduction concentration-MBRC-1,000 and 2,000 µg/ml) cultures. CO also inhibited biofilm formation (MBIC) at concentrations above 250 µg/ml for both species tested. However, synergism with amphotericin B was not observed. Thus, CO is a natural anticandidal agent that can be effectively utilised for the control of the yeasts tested.

New antimicrobial therapies used against fungi present in subgingival sites--a brief review.

Although the main reservoir of Candida spp. is believed to be the buccal mucosa, these microorganisms can coaggregate with bacteria in subgingival biofilm and adhere to epithelial cells. The treatment of periodontal disease includes scaling and root planning (SRP) associated with proper oral hygiene. However, some patients may have negative responses to different therapeutic procedures, with a continuous loss of insertion, so the use of antimicrobials is needed as an adjuvant to SRP treatment. The use of a broad-spectrum antibiotic, such as tetracycline and metronidazole, as an aid in periodontal treatment has also been a factor for the development of superinfections by resistant bacteria and Candida species, even in patients with HIV. In the dental practice, the most commonly used antifungals are nystatin and fluconazole. However, the introduction of new drugs like the next generation of azoles is essential before the onset of emergent species in periodontal disease. Plants are good options for obtaining a wide variety of drugs. This alternative could benefit a large population that uses plants as a first treatment option. Plants have been used in medicine for a long time and are extensively used in folk medicine, because they represent an economic alternative, are easily accessible and are applicable to various diseases. Herein, we briefly review the literature pertaining the presence of Candida sp. in periodontal pockets, the conventional antifungal resistance and new therapies that include natural antifungal agents are reviewed.

Enolase from Paracoccidioides brasiliensis: isolation and identification as a fibronectin-binding protein.

Paracoccidioides brasiliensis yeast cells can enter mammalian cells and may manipulate the host cell environment to favour their own growth and survival. Moreover, fibronectin and several other host extracellular matrix proteins are recognized by various components of the yeast cell extracts. The present study was designed to isolate and characterize a fibronectin-binding protein from P. brasiliensis. We also compared P. brasiliensis strain 18, tested before (Pb18a) and after (Pb18b) animal passage, in relation to its adhesion and invasion processes. Extracts from both samples, when cultured on blood agar solid medium, showed higher levels of protein expression than when the same samples were cultured on Fava-Netto solid medium, as demonstrated by two-dimensional electrophoresis and SDS-PAGE. Also, both Pb18a and Pb18b exhibited stronger adhesion to A549 epithelial cells when cultured on blood agar medium than when cultured on Fava-Netto medium. Ligand affinity binding assays revealed a protein of 54 kDa and pI 5.6 in P. brasiliensis cell-free extracts with the properties of a fibronectin-binding adhesin, which was characterized by tryptic digestion and mass spectroscopy as a homologue of enolase from P. brasiliensis. Antibody raised against this 54 kDa protein abolished 80 % of P. brasiliensis adhesion to A549 epithelial cells. Our results demonstrate that P. brasiliensis produces a fibronectin-binding adhesin, irrespective of the culture medium, and that this activity can be inhibited by a specific antibody and is involved in the adhesion of the fungus to pulmonary epithelial cells.

The use of standard methodology for determination of antifungal activity of natural products against medical yeasts Candida sp and Cryptococcus sp

Methodology for testing natural compounds for determination of antifungal activity had been developed with adaptations. The most used are bioautography and agar diffusion with a complex and no defined media. In this study, different methods for determination of antifungal activity of natural products are discussed and the use of M27-A2 microdilution test from CLSI (Clinical and Laboratory Standards Institute, 2002), as general standard methodology for testing plant extracts activity is recommended.

Metodologias para determinar atividade antifúngica foram desenvolvidas com adaptações para avaliar produtos naturais. As mais usadas são bioautografia e o método de difusão em agar, que empregam meios de cultura complexos, não definidos. Neste estudo são discutidos os métodos para determinação de atividade antifúngica de produtos naturais e é recomendado o uso do micrométodo modificado segundo o documento M27A2 do CLSI.

Molecular typing and antifungal susceptibility of clinical sequential isolates of Cryptococcus neoformans from Sao Paulo State, Brazil.

The antifungal susceptibility profiles and the genetic variability of 83 sequential clinical isolates of Cryptococcus neoformans, including four Cryptococcus gattii isolates, obtained from 38 Sao Paulo AIDS patients with cryptococcal meningitis were assessed by electrophoretic karyotyping and random amplified polymorphic DNA (RAPD) analysis. The majority of the Cryptococcus neoformans isolates were highly susceptible to amphotericin B and fluconazole. Twenty percent of the minimum inhibitory concentration values for amphotericin B varied from 0.5 to 1 micro g mL(-1). For fluconazole, 22% occurred in the range 8-16 mug mL(-1). Sequential isolates from nine patients showed a trend towards lower susceptibility to fluconazole, flucytosine, itraconazole and amphotericin B. The results of molecular typing by electrophoretic karyotyping and RAPD analysis showed the presence of 22 electrophoretic karyotypes (EK) and 15 RAPD profiles that were highly correlated. Our results provided evidence for the occurrence of genetic changes in some strains associated with microevolution during the course of infection. We also observed both microevolution and simultaneous coinfection with two distinct Cryptococcus neoformans strains in one patient. In some patients, we found changed EK- and RAPD patterns in association with increased MIC values.

Strain differentiation of Trichophyton rubrum by randomly amplified polymorphic DNA and analysis of rDNA nontranscribed spacer.

Trichophyton rubrum is the most common pathogen causing dermatophytosis. Molecular strain-typing methods have recently been developed to tackle epidemiological questions and the problem of relapse following treatment. A total of 67 strains of T. rubrum were screened for genetic variation by randomly amplified polymorphic DNA (RAPD) analysis, with two primers, 5'-d[GGTGCGGGAA]-3' and 5'-d[CCCGTCAGCA]-3', as well as by subrepeat element analysis of the nontranscribed spacer of rDNA, using the repetitive subelements TRS-1 and TRS-2. A total of 12 individual patterns were recognized with the first primer and 11 with the second. Phylogenetic analysis of the RAPD products showed a high degree of similarity (>90 %) among the epidemiologically related clinical isolates, while the other strains possessed 60 % similarity. Specific amplification of TRS-1 produced three strain-characteristic banding patterns (PCR types); simple patterns representing one copy of TRS-1 and two copies of TRS-2 accounted for around 85 % of all isolates. It is concluded that molecular analysis has important implications for epidemiological studies, and RAPD analysis is especially suitable for molecular typing in T. rubrum.