Rachel Basques Caligiorne

Species Diversity and Polymorphism in the Exophiala spinifera Clade Containing Opportunistic Black Yeast-Like Fungi

ABSTRACT A monophyletic group of black yeast-like fungi containing opportunistic pathogens around Exophiala spinifera is analyzed using sequences of the small-subunit (SSU) and internal transcribed spacer (ITS) domains of ribosomal DNA. The group contains yeast-like and annellidic species (anamorph genus Exophiala) in addition to sympodial taxa (anamorph genera Ramichloridium and Rhinocladiella). The new species Exophiala oligosperma, Ramichloridium basitonum, and Rhinocladiella similis are introduced and compared with their morphologically similar counterparts at larger phylogenetic distances outside the E. spinifera clade. Exophiala jeanselmei is redefined. New combinations are proposed in Exophiala: Exophiala exophialae for Phaeococcomyces exophialae and Exophiala heteromorpha for E. jeanselmei var. heteromorpha.

Combined diagnostic methods identify a remarkable proportion of asymptomatic Leishmania (Leishmania) chagasi carriers who present modulated cytokine profiles

Peripheral blood samples of 138 co-habitants from 25 families with recently diagnosed cases of visceral leishmaniasis in the Metropolitan Region of Belo Horizonte, Minas Gerais, Brazil, were analyzed by indirect fluorescent antibody test (IFAT), rK39 and Leishmania chagasi Enzyme Linked Immunosorbent Assay (ELISA), intradermal skin-test and Polymerase Chain Reaction (PCR) over a 12-month period. The cumulative positivity was significantly higher by PCR (29.7%) than by IFAT, rK39 ELISA, L. chagasi ELISA and intradermal skin-test (5.1%, 6.5%, 14.5% and 2.9%, respectively). In addition, the cytokine profile was measured in 16 of the 138 volunteers, of whom eight were asymptomatic carriers and eight were non-infected co-habitants. The innate immunity cells from asymptomatic carriers displayed, upon in vitro antigenic stimulation, a modulated increase in cytokine synthesis that was distinct from that observed in non-infected volunteers. This study suggests that the identification of a large proportion of asymptomatic carriers is facilitated when more than one diagnostic method is applied and that a mixed pattern of immune response is correlated with clinical status of asymptomatic individuals. These observations suggest also that asymptomatic infection by L. chagasi is a frequent event and that control programs could benefit by including this indicator in their interventions.

Leishmanicidal, trypanocidal, and cytotoxic activities of endophytic fungi associated with bioactive plants in Brazil

One hundred and twenty-one isolates of endophytic fungi were recovered from leaves of the bioactive Brazilian plant species Ageratum myriadenia, Palicourea tetraphylla, Piptadenia adiantoides, and Trixis vauthieri. All fungal isolates were cultivated in liquid media and crude extracts were obtained with ethyl acetate. The crude extracts were tested in bioassay panels using Leishmania amazonensis, Trypanosoma cruzi, the enzyme trypanothione reductase (TryR) from Trypanosoma cruzi, and three human cancer cell lines. Thirty-three extracts (27.2%) exhibited at least one biological activity. Seventeen extracts (14%) were cytotoxic against one or more human cancer cell line with the IC50 values ranged of >0.2 to 25 µg/mL. Twenty-four extracts (19.8%) inhibited the activity of TryR, and three showed ability to inhibit the growth of T. cruzi above 60% and their IC50 values ranged among 1 to 10 µg/mL. Eleven extracts (9%) were able to inhibit the growth of L. amazonensis and showed with IC50 values ranged among 4.6 to 24.4 µg/mL. The endophytic fungi were identified as belonging to the genera Alternaria, Arthrinium, Cochliobolus, Colletotrichum, Penicillium, Fusarium, and Gibberella. An interesting result was obtained for the bioactive isolates UFMGCB 508, 537, 899 and 903, which were related to fungi associated with medicinal plants native to Asia, Australia, Africa, and Polynesia. These results indicate that bioactive plants living in Brazilian ecosystems are a potential host of endophytic fungi able to produce bioactive prototype molecules for drug development against neglected tropical diseases.

Leishmanicidal Metabolites from Cochliobolus sp., an Endophytic Fungus Isolated from Piptadenia adiantoides (Fabaceae)

Protozoan parasites belonging to genera Leishmania and Trypanosoma are the etiological agents of severe neglected tropical diseases (NTDs) that cause enormous social and economic impact in many countries of tropical and sub-tropical areas of the world. In our screening program for new drug leads from natural sources, we found that the crude extract of the endophytic fungus Cochliobolus sp. (UFMGCB-555) could kill 90% of the amastigote-like forms of Leishmania amazonensis and inhibit by 100% Ellmans reagent reduction in the trypanothione reductase (TryR) assay, when tested at 20 µg mL−1. UFMGCB-555 was isolated from the plant Piptadenia adiantoides J.F. Macbr (Fabaceae) and identified based on the sequence of the internally transcribed spacer (ITS) regions of its ribosomal DNA. The chromatographic fractionation of the extract was guided by the TryR assay and resulted in the isolation of cochlioquinone A and isocochlioquinone A. Both compounds were active in the assay with L. amazonensis, disclosing EC50 values (effective concentrations required to kill 50% of the parasite) of 1.7 µM (95% confidence interval = 1.6 to 1.9 µM) and 4.1 µM (95% confidence interval = 3.6 to 4.7 µM), respectively. These compounds were not active against three human cancer cell lines (MCF-7, TK-10, and UACC-62), indicating some degree of selectivity towards the parasites. These results suggest that cochlioquinones are attractive lead compounds that deserve further investigation aiming at developing new drugs to treat leishmaniasis. The findings also reinforce the role of endophytic fungi as an important source of compounds with potential to enter the pipeline for drug development against NTDs.

Altenusin, a biphenyl isolated from the endophytic fungus Alternaria sp., inhibits trypanothione reductase from Trypanosoma cruzi

Parasitic protozoan species belonging to the genera Trypanosoma and Leishmania are the etiological agents of several diseases in tropical areas of the world, for which there is an urgent need for effective and affordable treatment. In this regard, we are screening the Brazilian biodiversity, especially its flora and mycota, for natural products that could serve as leads for drug development against these diseases. Trypanothione reductase (TR) is an enzyme involved in the protection of Trypanosoma and Leishmania species against oxidative stress, and is considered to be a validated drug target. The endophytic fungus Alternaria sp. (UFMGCB55) was isolated from the plant Trixis vauthieri DC (Asteraceae), known to contain trypanocidal compounds. The organic extract of the culture of Alternaria sp. was able to inhibit TR by 99%, when tested at 20 microg mL(-1). Fractionation of the extract identified altenusin, a biphenyl derivative with an IC50 value of 4.3+/-0.3 microM in the TR assay. This compound is the first in its class to have shown TR inhibitory activity, opening new perspectives for the design of more effective derivatives that could serve as drug leads for new chemotherapeutic agents to treat trypanosomiasis and leishmaniasis.

Lesion aspirate culture for the diagnosis and isolation of Leishmania spp. from patients with cutaneous leishmaniasis.

The detection of Leishmania spp. in skin lesion aspirates, using a puncture technique, was evaluated in 76 patients with cutaneous leishmaniasis (CL) who were referred to a Leishmaniasis Reference Centre in Brazil. CL was defined based on skin lesions suggestive of the disease and on a positive result of the Montenegro skin test or Giemsa-stained imprints of biopsy fragments. The aspirates were cultured using a vacuum tube device containing culture medium and evaluated for the presence of Leishmania spp. The biphasic medium culture was examined once a week for three weeks. Promastigotes were observed in 53/76 (69.7%) cultures. Stained smears from 60 of the 76 patients were evaluated using PCR-RFLP to detect the conserved minicircle region of Leishmania spp. and to classify the parasite. Of these patients, 45 (75%) showed positive results in aspirate culture and 15 presented negative results. The PCR was positive in 80% (53/60) samples. The PCR-RFLP profile was determined in 49 samples, of which 45 (92%) showed a pattern compatible with Leishmania (Viannia) braziliensis. The aspirate culture is a sensitive and feasible method for diagnosing CL and may be routinely adopted by health services for L. (V.) braziliensis isolation and identification.

Internal Transcribed Spacer rRNA Gene-Based Phylogenetic Reconstruction Using Algorithms with Local and Global Sequence Alignment for Black Yeasts and Their Relatives

ABSTRACT Sequences of rRNA gene internal transcribed spacer (ITS) of a standard set of black yeast-like fungal pathogens were compared using two methods: local and global alignments. The latter is based on DNA-walk divergence analysis. This method has become recently available as an algorithm (DNAWD program) which converts sequences into three-dimensional walks. The walks are compared with, or fit to, each other generating global alignments. The DNA-walk geometry defines a proper metric used to create a distance matrix appropriated for phylogenetic reconstruction. In this work, the analyses were carried out for species currently classified in Capronia, Cladophialophora, Exophiala, Fonsecaea, Phialophora, and Ramichloridium. Main groups were verified by small-subunit rRNA gene data. DNAWD applied to ITS2 alone enabled species recognition as well as phylogenetic reconstruction reflecting clades discriminated in small-subunit rRNA gene phylogeny, which was not possible with any other algorithm using local alignment for the same data set. It is concluded that DNAWD provides rapid insight into broader relationships between groups using genes that otherwise would be hardly usable for this purpose.

Antifungal activity of trichothecenes from Fusarium sp. against clinical isolates of Paracoccidioides brasiliensis

Paracoccidioidomycosis (PCM), a human mycosis caused by the dimorphic fungus Paracoccidioides brasiliensis, is a serious public health problem in several countries of Latin America. In our search we found that the crude extract of the endophytic fungus UFMGCB 551 was able to inhibit several clinical strains of P. brasiliensis, and was also active in the bioautographic assay against Cladosporium sphaerospermum. The endophytic fungus UFMGCB 551 was isolated from the plant Piptadenia adiantoides J.F. Macbr (Fabaceae). The fungus was identified as Fusarium sp. based on its macro‐ and micro‐morphology, and on the sequence of the internally transcribed spacer regions (ITS) of its rRNA gene. The chromatographic fractionation of the fungal extract was guided by the bioautographic assay to afford three known trichothecene mycotoxins: T2‐toxin (1) and a mixture of 8‐n‐butyrylneosolaniol (2) and 8‐isobutyrylsolaniol (3). The minimal inhibitory concentrations (MIC) of the these compounds against eleven clinical strains of P. brasiliensis were evaluated and found to be in the range between 75 and 640 nmol l−1 for 1 and 160–640 nmol l−1 for the mixture of 2 and 3.

Detection of Leishmania kDNA in human serum samples for the diagnosis of visceral leishmaniasis

The performance of PCR to detect Leishmania kDNA in serum for the diagnosis of visceral leishmaniasis (VL) was assessed in serum samples from 65 patients with VL, 17 non-infected individuals and 17 patients with other febrile hepatosplenic diseases. Serum PCR showed a sensitivity of 85%, specificity of 100% and efficiency of 90%. The sensitivity values obtained for blood PCR (97%) and rK39 ELISA (95%) were significantly higher (P=0.01) than the values observed for L. chagasi ELISA (88%) and serum PCR (85%), whilst no difference was observed among the specificity rates obtained with rK39 ELISA (94%; P=0.47) and L. chagasi ELISA (85%; P=0.06). This work suggests that the use of serum samples may be an alternative for the diagnosis of VL when peripheral blood samples are not available or require significant operational efforts.

Histoplasmosis outbreak in Tamboril cave—Minas Gerais state, Brazil

In this study, we report four cases of Histoplasma capsulatum infection in eight biologists who made an expedition to determine the prevalence of this fungus in a cave localized in the northwest of Minas Gerais state, Brazil. This case report demonstrates the importance of evaluating the H. capsulatum presence in Brazilian caves before opening to public visitations.