Randomly Amplified Polymorphic DNA as a Valuable Tool for Epidemiological Studies of Paracoccidioides brasiliensis

Abstract

Randomly amplified polymorphic DNA (RAPD) has been successfully used to detect genetic variations among isolates of Paracoccidioides brasiliensis. However, the usefulness of this technique for assessing important parasitic properties is still unconfirmed. In the present work we further investigated the applicability of RAPD in revealing important intrinsic and extrinsic features of this fungus associated with geographical origin, time of isolation, source of clinical specimen, clinical forms of human disease and also in vitro and in vivo susceptibility to antimicrobial and antifungal drugs. The RAPD patterns allowed us to distinguish all of the analyzed strains, which included 26 clinical isolates, 2 animal isolates, and 1 environmental isolate of P. brasiliensis obtained from different geographic regions, confirming the strong discriminating power of this technique. A phenetic tree, build from the RAPD data, showed that although the two nonclinical Brazilian strains were set together the majority of the clinical Brazilian strains were randomly distributed through different sub-branches of a major cluster without any correlation to any of the parameters analyzed. A second major cluster, however, has grouped isolates from Mato Grosso and Roraima (Brazil) that not only were susceptible in vitro to trimethoprim-sulfamethoxazole but also produced a good in vivo response. These results open new vistas for epidemiological and clinical studies of P. brasiliensis.Paracoccidioides brasiliensis is a dimorphic fungus alternating between the mycelial form observed in room temperatures and the yeast form found in infected tissues or in culture at 35 to 37°C. The human paracoccidioidomycosis caused by this fungus is characterized by a range of clinical manifestations from benign or asymptomatic forms to severe and disseminated disease that is often fatal. The exact causes of the clinical pleomorphism are not understood, but it is reasonable to presume that factors associated with both the fungus and the patient are involved. In relation to the fungus, although wide biological and biochemical diversity has been observed, no clear correlation with pathogenesis has been demonstrated (3,9,12,13,17,20,23). Until now very little has been known about the genome sequences of P. brasiliensis; hence, the use of molecular approaches for isolate typing has been difficult.The development of the randomly amplified polymorphic DNA (RAPD) technique represented a landmark in the molecular characterization of diverse organisms, especially for microbial strain identifications when very few genomic sequences are available. In relation to other molecular techniques, RAPD analysis offers the advantage of requiring small amounts of DNA, simpler procedures, and the use of arbitrary primers (26). Due to these characteristics, RAPD has been widely used for human diseases to detect genomic variations among isolates of fungi such as Histoplasma capsulatum (27), Cryptococcus neoformans (19), Aspergillus fumigatus (1), and P. brasiliensis (3,9,12,13,23). ...

Molecular characterisation of Sporothrix schenckii isolates from humans and cats involved in the sporotrichosis epidemic in Rio de Janeiro, Brazil

“…The band pattern of the US strain was totally different from those of Brazil, which is in accordance with results from other studies of S. schenckii, as well as with other pathogenic fungi, such as Paracoccidioides brasiliensis and Histoplasma capsulatum (Muniz et al 2001, Mesa-Arango et al 2002, Hahn et al 2003. However, the genetic variability or polymorphic patterns found in different geographical areas can also be due to different ecological processes or genetic factors that promote these polymorphisms (Mesa-Arango et al 2002).…”

Diversity in Paracoccidioides brasiliensis . The PbGP43 gene as a genetic marker

“…Some authors [52,54] have mentioned that they could not discriminate isolates by their geographical localization using similar primers as Calcagno et al [55]. However, their results are difficult to compare, because in those investigations either the isolates analyzed were 80% from Brazil [54] or geographically restricted to São Paulo [52]. Hence, the intrinsic genetic diversity within each group prevailed.…”

“…Geographic discrimination of the same samples was later confirmed using restriction fragment length polymorphism (RFLP), where the restriction enzymes HinfI and HincII produced clear RFLP patterns [56]. Some authors [52,54] have mentioned that they could not discriminate isolates by their geographical localization using similar primers as Calcagno et al [55]. However, their results are difficult to compare, because in those investigations either the isolates analyzed were 80% from Brazil [54] or geographically restricted to São Paulo [52].…”

“…Hahn et al [54] tested 26 clinical, one armadillo and two soil isolates in their RAPD analysis with 5 primers, which individually generated similar phenograms with a subdivided group I and small group II composed almost exclusively of isolates from Mato Grosso, Brazil. The authors could not correlate genotypic groups with any of the patients' features.…”

“…Group I isolates showed regressive pattern of infection, with little tissue damage and dissemination, while infection with group II isolates (Pb01 and 7455) resulted in intense, progressive and disseminated peritonitis, with extensive tissue damage in the diaphragm and other organs caused by numerous non-organized granuloma. Other reports claimed unsuccessful correlation of RAPD genetic groups with virulence or PCM clinical features [53][54][55], however they generally relied on medical records and/or literature data about the isolate degrees of virulence. More recently, Kurokawa et al [52] tested virulence of RAPD genetically distinct isolates and could not correlate the results; however they used outbred swiss mice.…”