Methodological development and biological observations of cell free DNA with a simple direct fluorescent assay in colorectal cancer.

BACKGROUND: Cell free DNA (cfDNA) has shown promising utility as prognostic biomarker for patients with colorectal cancer (CRC), with an ongoing need to optimize and validate the laboratory methodology. Here, we report our optimization and validation of a direct fluorescent assay and display the potential utility in patients with colorectal cancer. METHODS: Plasma cfDNA was analyzed by a direct fluorescent assay (DFA) and compared to quantification by droplet digital PCR (ddPCR). For clinical validation, baseline blood samples were available for a total of 273 patients from six different Nordic trials, covering patients with locally advanced rectal cancer (n = 176, cohorts A + B), liver limited metastatic CRC (n = 75C + D) and wide spread metastatic CRC (n = 22 E + F). RESULTS: Validating the DFA analysis with ddPCR revealed a strong correlation with an R2 of 0.81. For the clinical cohorts, the levels of cfDNA were: 0.8 ng/uL (95%CI 0.75-0.83) (A + B), 0.93 ng/uL (95%CI 0.86-1.02) (C + D) and 1.2 ng/uL (95%CI 0.85-1.47) (E + F), respectively (p < 0.01). All cohorts of colorectal cancer had higher levels of cell free DNA than healthy individuals (n = 94) (p < 0.01). CONCLUSION: Analysis of cell free DNA by a direct fluorescent assay could be an attractive laboratory option for a rapid inexpensive quantification of cell free DNA.

Circulating cell-free DNA as predictor of treatment failure after neoadjuvant chemo-radiotherapy before surgery in patients with locally advanced rectal cancer.

Background: Treatment of patients with locally advanced rectal cancer (LARC) is based on a combination of chemo-radiotherapy (CRT) and surgery. The rate of distant recurrences remains over 25%. Circulating cell-free DNA (cfDNA) in plasma is a mixture of normal and cancer-specific DNA segments and is a promising biomarker in patients with colorectal cancer. The aim of our study was to investigate plasma cfDNA as a prognostic marker for outcome in patients with LARC treated with neoadjuvant CRT and surgery. Patients and methods: In total, 123 patients with LARC were included in 2 biomarker studies. Patients were treated with neoadjuvant CRT before TME surgery. Fifty-two (42%) of the patients received induction chemotherapy with capecitabine + oxaliplatin. Total cfDNA was measured by direct fluorescent assay in EDTA plasma samples obtained at baseline, after induction chemotherapy, and after CRT. Serial samples 5 years after surgery were collected in 51 patients (41%). Results: Median follow-up was 55 months. Distant or local recurrence was seen in 30.9% of the patients. Patients with baseline cfDNA levels above the 75th quartile had a higher risk of local or distant recurrence and shorter time to recurrence compared with patients with plasma cfDNA below the 75th percentile (HR = 2.48, 95% CI: 1.3-4.8, P = 0.007). The same applied to disease-free survival (DFS) (HR = 2.43, 95% CI: 1.27-4.7, P = 0.015). In multivariate analysis, a high cfDNA level was significantly associated with time to progression and DFS. During follow-up, the association remained significant regardless of time point for sample analysis. Conclusion: We have demonstrated an association between a high baseline plasma level of cfDNA and increased risk of recurrence, shorter time to recurrence, and shorter DFS in patients with LARC. Consequently, cfDNA could potentially improve pre- and post-treatment risk assessment and facilitate individualized therapy for patients with LARC.

Comparação entre as medidas morfométricas de equinos Mangalarga Marchador de marcha batida e marcha picada / Comparison between the morphometric measures of Mangalarga Marchador horses of batida marcha and picada marcha

The present study aimed to compare the morphometric measurements of Mangalarga Marchador horses of batida and picada marcha. Twenty-two linear and eight angular measurements of 222 males (130 of batida marcha and 92 of picada marcha) and 266 females (168 of batida marcha and 98 picada marcha) were compared in a completely randomized design, consisting of two treatments: horses of batida and picada marcha. The results were submitted to analysis of variance and means were compared by Fisher test (P<0.05). It was concluded that most of the measures of Mangalarga Marchador horses of batida and picada marcha have similar values, however, there are differences between some angles of members...

Isolation of Cryptococcus neoformans and Cryptococcus gattii from trunk hollows of living trees in Buenos Aires City, Argentina.

The aim of this work was to describe the distribution of the members of the Cryptococcus species complex (Cryptococcus neoformans and C. gattii) in hollows of trees from seven parks in Buenos Aires City, to determine the serotypes and genotypes of these environmental isolates and to compare them with the ones reported in the 2001 survey. Four hundred and eighty nine samples were collected by swabbing all trees which had hollows or fissures in the seven parks studied. Each tree was sampled once during the study period and one or more isolates were recovered from each swab. Eight isolates of C. neoformans and 18 isolates of C. gattii were recovered from 15 out of 489 tree samples. C. neoformans was isolated from Tipuana tipu, Eucalyptus spp., and Phoenix sp. All isolates were serotype A and belonged to genotype VNI. C. gattii was isolated from Tipuana tipu, Cedrus deodara, Eucalyptus spp., Acacia visca, Cupresus sempervirens and Ulmus campestrus. All isolates were serotype B and genotype VGI, like both C. gattii strains isolated in 2001. On two occasions, both species were isolated from the same tree sample. These results reinforce and extend our previous findings especially about the presence of C. gattii serotype B, genotype VGI, in Argentina.

[1st environmental isolation of Cryptococcus neoformans var. gattii in Argentina]. / Primer aislamiento ambiental de Cryptococcus neoformans var. gattii en Argentina.

Two strains of C. neoformans var. gattii serotype B were isolated from a park in Buenos Aires city, Argentina. Samples were collected in spring, rubbing with swabs on the inner hollow of Eucalyptus spp. and other trees, which were not identified at the collection moment. Samples were swabbed directly onto cafeic acid with antibiotic agar, then incubated at 28 degrees C and observed daily for a week. Isolates were recovered from cafeic acid medium showing characteristic colonies of the species that allowed differentiation from other microorganisms of the sample. Identification was performed using carbohydrate fermentation, assimilation of nitrogen and carbon sources, and urease and phenoloxidase detection. Strain variety was determined with canavanine-glicine-bromotimol blue agar medium (CGB). Genotypic characterization using AP-PCR with (GACA)4 aleatory primer showed that both band profiles could be differentiated by only one band. These two strains were isolated from tree species different from Eucalyptus spp. These isolates from natural sources demonstrate that C. neoformans var. gattii efosts in Buenos Aires city environment and that it is not necessarily associated with Eucalyptus spp. The presence of C. neoformansvar. gattii in the environment, detected for the first time in Argentina, may explain its sporadic association with human infections in the local population.

Primer aislamiento ambiental de Cryptococcus neoformans var. gattii en Argentina / Firs environmental detection of Cryptococcus neoformans var. gattii in Argentina

Dos cepas de C.neoformans var. gattii serotipo B, fueron aisladas en el Parque de los Patricios de la ciudad de Buenos Aires, Argentina. Las muestras se recogieron en primavera, mediante el hisopado de la superficie interior de huecos y grietas de Eucalyptus spp. y otros árboles que, lamentablemente, no fueron identificados en el momento de la recolección. Los hisopos se inocularon en placas de agar ácido cafeico suplementado con antibióticos que se incubaron a 28§C durante una semana con observación diaria. Los aislamientos produjeron colonias características de la especie, lo que permitió detectarlos y separarlos de otros microorganismos presentes en la muestra. La identificación se realizó mediante estudios de fermentación de carbohidratos, asimilación de fuentes de carbono y nitrógeno y detección de ureasa y fenoloxidasa. La variedad se determinó utilizando el medio agarizado de canavanina-glicina-azul de bromotimol (CGB). La caracterización genotípica por reacción en cadena de la polimerasa con primer aleatorio (AP-PCR con el primer (GACA)4)mostró que el patrón de bandas de ambos aislamientos se diferenciaba en una única banda. En ambos casos el material provino de especies arbóreas diferentes de Eucalyptus spp. Estos dos aislamientos de fuentes naturales demuestran que C.neoformans var. gattii está presente en el medio ambiente de la ciudad de Buenos Aires, no necesariamente asociado a Eucalyptus spp. Este hallazgo podría explicar su asociación esporádica a infecciones humanas en la población local.

Primer aislamiento ambiental de Cryptococcus neoformans var. gattii en Argentina / Firs environmental detection of Cryptococcus neoformans var. gattii in Argentina

Dos cepas de C.neoformans var. gattii serotipo B, fueron aisladas en el Parque de los Patricios de la ciudad de Buenos Aires, Argentina. Las muestras se recogieron en primavera, mediante el hisopado de la superficie interior de huecos y grietas de Eucalyptus spp. y otros árboles que, lamentablemente, no fueron identificados en el momento de la recolección. Los hisopos se inocularon en placas de agar ácido cafeico suplementado con antibióticos que se incubaron a 28ºC durante una semana con observación diaria. Los aislamientos produjeron colonias características de la especie, lo que permitió detectarlos y separarlos de otros microorganismos presentes en la muestra. La identificación se realizó mediante estudios de f

Primer aislamiento ambiental de Cryptococcus neoformans var. gattii en Argentina. / [1st environmental isolation of Cryptococcus neoformans var. gattii in Argentina]

Two strains of C. neoformans var. gattii serotype B were isolated from a park in Buenos Aires city, Argentina. Samples were collected in spring, rubbing with swabs on the inner hollow of Eucalyptus spp. and other trees, which were not identified at the collection moment. Samples were swabbed directly onto cafeic acid with antibiotic agar, then incubated at 28 degrees C and observed daily for a week. Isolates were recovered from cafeic acid medium showing characteristic colonies of the species that allowed differentiation from other microorganisms of the sample. Identification was performed using carbohydrate fermentation, assimilation of nitrogen and carbon sources, and urease and phenoloxidase detection. Strain variety was determined with canavanine-glicine-bromotimol blue agar medium (CGB). Genotypic characterization using AP-PCR with (GACA)4 aleatory primer showed that both band profiles could be differentiated by only one band. These two strains were isolated from tree species different from Eucalyptus spp. These isolates from natural sources demonstrate that C. neoformans var. gattii efosts in Buenos Aires city environment and that it is not necessarily associated with Eucalyptus spp. The presence of C. neoformansvar. gattii in the environment, detected for the first time in Argentina, may explain its sporadic association with human infections in the local population.

[Molecular characterization of autochthonous clinical isolates of Histoplasma capsulatum var. capsulatum using RAPD-PCR]. / Caracterización molecular de aislamientos clínicos autóctonos de Histoplasma capsulatum var. capsulatum mediante RAPD-PCR.

The aim of our work was to determine the genotypic relatedness among H. capsulatum isolates from Argentinian patients with or without AIDS and isolates from other sources and geographic origins. We analyzed band patterns of 16 isolates obtained by RAPD-PCR, 11 Argentinian clinical isolates (8 from patients with AIDS and 3 from patients without AIDS), 2 strains from the CDC, 2 isolates from Mexico and one isolate from Uruguay. With the band patterns obtained, we built a dendrogram, which showed 3 major groups. Group I contained 2 isolates from Mexico, group II 1 strain from CDC and group III included 13 isolates, and was divided into 4 subgroups. Subgroup IIIa contained 2 isolates from patients with and without AIDS. Subgroup IIIb included isolates from patients without AIDS from Chaco. Subgroup IIIc contained 7 isolates from Argentina (from Buenos Aires province) and the Uruguayan isolate (6 isolates from patients with AIDS). Subgroup IIId included only one strain from CDC. All the Argentinian and the Uruguayan isolates went into group III, subgroups IIIa, IIIb and IIIc. This is a preliminary study; however, a relatedness of the RAPD-PCR band patterns and the geographic origin was observed. Although there might be a probable association between immunocompromised patients with AIDS and infective capability of some H. capsulatum isolates, further studies with a larger number of isolates from different sources and geographic origins are necessary to confirm these results.

Caracterización molecular de aislamientos clínicos autóctonos de Histoplasma capsulatum var. capsulatum mediante RAPD-PCR / Molecular characterization of autochthonous clinical isolates of Histoplasma capsulatum var. capsulatum using RAPD-PCR

The aim of our work was to determine the genotypic relatedness among H. capsulatum isolates from Argentinian patients with or without AIDS and isolates from other sources and geographic origins. We analyzed band patterns of 16 isolates obtained by RAPD-PCR, 11 Argentinian clinical isolates (8 from patients with AIDS and 3 from patients without AIDS), 2 strains from the CDC, 2 isolates from Mexico and one isolate from Uruguay. With the band patterns obtained, we built a dendrogram, which showed 3 major groups. Group I contained 2 isolates from Mexico, group II 1 strain from CDC and group III included 13 isolates, and was divided into 4 subgroups. Subgroup IIIa contained 2 isolates from patients with and without AIDS. Subgroup IIIb included isolates from patients without AIDS from Chaco. Subgroup IIIc contained 7 isolates from Argentina (from Buenos Aires province) and the Uruguayan isolate (6 isolates from patients with AIDS). Subgroup IIId included only one strain from CDC. All the Argentinian and the Uruguayan isolates went into group III, subgroups IIIa, IIIb and IIIc. This is a preliminary study; however, a relatedness of the RAPD-PCR band patterns and the geographic origin was observed. Although there might be a probable association between immunocompromised patients with AIDS and infective capability of some H. capsulatum isolates, further studies with a larger number of isolates from different sources and geographic origins are necessary to confirm these results.

Caracterización molecular de aislamientos clínicos autóctonos de Histoplasma capsulatum var. capsulatum mediante RAPD-PCR / Molecular characterization of autochthonous clinical isolates of Histoplasma capsulatum var. capsulatum using RAPD-PCR

The aim of our work was to determine the genotypic relatedness among H. capsulatum isolates from Argentinian patients with or without AIDS and isolates from other sources and geographic origins. We analyzed band patterns of 16 isolates obtained by RAPD-PCR, 11 Argentinian clinical isolates (8 from patients with AIDS and 3 from patients without AIDS), 2 strains from the CDC, 2 isolates from Mexico and one isolate from Uruguay. With the band patterns obtained, we built a dendrogram, which showed 3 major groups. Group I contained 2 isolates from Mexico, group II 1 strain from CDC and group III included 13 isolates, and was divided into 4 subgroups. Subgroup IIIa contained 2 isolates from patients with and without AIDS. Subgroup IIIb included isolates from patients without AIDS from Chaco. Subgroup IIIc contained 7 isolates from Argentina (from Buenos Aires province) and the Uruguayan isolate (6 isolates from patients with AIDS). Subgroup IIId included only one strain from CDC. All the Argentinian and the Uruguayan isolates went into group III, subgroups IIIa, IIIb and IIIc. This is a preliminary study; however, a relatedness of the RAPD-PCR band patterns and the geographic origin was observed. Although there might be a probable association between immunocompromised patients with AIDS and infective capability of some H. capsulatum isolates, further studies with a larger number of isolates from different sources and geographic origins are necessary to confirm these results.(AU)

Maxillary sinusitis caused by Actinomucor elegans.

We report the first case of maxillary sinusitis caused by Actinomucor elegans in an 11-year-old patient. Histopathological and mycological examinations of surgical maxillary sinuses samples showed coenocytic hyphae characteristic of mucoraceous fungi. The fungi recovered had stolons and rhizoids, nonapophyseal and globose sporangia, and whorled branched sporangiophores and was identified as A. elegans. After surgical cleaning and chemotherapy with amphotericin B administered intravenously and by irrigation, the patient became asymptomatic and the mycological study results were negative.

Caracterización molecular de aislamientos clínicos autóctonos de Histoplasma capsulatum var. capsulatum mediante RAPD-PCR. / [Molecular characterization of autochthonous clinical isolates of Histoplasma capsulatum var. capsulatum using RAPD-PCR]

The aim of our work was to determine the genotypic relatedness among H. capsulatum isolates from Argentinian patients with or without AIDS and isolates from other sources and geographic origins. We analyzed band patterns of 16 isolates obtained by RAPD-PCR, 11 Argentinian clinical isolates (8 from patients with AIDS and 3 from patients without AIDS), 2 strains from the CDC, 2 isolates from Mexico and one isolate from Uruguay. With the band patterns obtained, we built a dendrogram, which showed 3 major groups. Group I contained 2 isolates from Mexico, group II 1 strain from CDC and group III included 13 isolates, and was divided into 4 subgroups. Subgroup IIIa contained 2 isolates from patients with and without AIDS. Subgroup IIIb included isolates from patients without AIDS from Chaco. Subgroup IIIc contained 7 isolates from Argentina (from Buenos Aires province) and the Uruguayan isolate (6 isolates from patients with AIDS). Subgroup IIId included only one strain from CDC. All the Argentinian and the Uruguayan isolates went into group III, subgroups IIIa, IIIb and IIIc. This is a preliminary study; however, a relatedness of the RAPD-PCR band patterns and the geographic origin was observed. Although there might be a probable association between immunocompromised patients with AIDS and infective capability of some H. capsulatum isolates, further studies with a larger number of isolates from different sources and geographic origins are necessary to confirm these results.

Estudio multicéntrico de micosis superficiales en Argentina. Grupo EMMS / Multicenter study of superficial mycoses in Argentina. EMMS Group

Dermatomycoses are an important sanitary problem. Our country counts with partial epidemiological data only referred to a few geographical areas. The aim of our work was to perform a multicenter study with the participation of 15 medical centers located at the Capital City and in 11 Provinces of Argentina with different socioeconomical and cultural characteristics. Between October and December 1998, 851 ambulatory patients of both sexes (58 female, 42 male) with lesions compatible with mycoses in skin, nails or scalp were studied. Only 51.6 of the patients had mycoses confirmed by direct microscopic examination and/ or culture. Dermatophytes caused 69.3 of the diagnosticated infections, yeast 12.2 and Malassezia furfur 13.3. Agents of dermatomycoses most frequently isolated were T. rubrum (52.7), T. mentagrophytes (19.2) and M. canis (14.2). C. albicans was the predominant causative agent of yeast infections (44.4). In children below 11 years old, the most frequent infections were tinea capitis 55.4 (M. canis 60, T. tonsurans and M. gypseum 8.3) and tinea corporis 20 (M. canis 46, T. mentagrophytes 30). The most frequent localizations in adult were toenail and feet (63) (T. rubrum 47.3 and 45.7, T. mentagrophytes 9.5 and 23.4, respectively), fingernail 15.6 (C. albicans 25 and T. rubrum 8.3) and trunk and extremities 12.2 (M. furfur 57.5 and T. mentagrophytes 14.9). Direct examination allowed the diagnosis of 94.66 of all positive samples and 91.74 of the cases with positive isolation.

Estudio multicéntrico de micosis superficiales en Argentina. Grupo EMMS / Multicenter study of superficial mycoses in Argentina. EMMS Group

Dermatomycoses are an important sanitary problem. Our country counts with partial epidemiological data only referred to a few geographical areas. The aim of our work was to perform a multicenter study with the participation of 15 medical centers located at the Capital City and in 11 Provinces of Argentina with different socioeconomical and cultural characteristics. Between October and December 1998, 851 ambulatory patients of both sexes (58 female, 42 male) with lesions compatible with mycoses in skin, nails or scalp were studied. Only 51.6 of the patients had mycoses confirmed by direct microscopic examination and/ or culture. Dermatophytes caused 69.3 of the diagnosticated infections, yeast 12.2 and Malassezia furfur 13.3. Agents of dermatomycoses most frequently isolated were T. rubrum (52.7), T. mentagrophytes (19.2) and M. canis (14.2). C. albicans was the predominant causative agent of yeast infections (44.4). In children below 11 years old, the most frequent infections were tinea capitis 55.4 (M. canis 60, T. tonsurans and M. gypseum 8.3) and tinea corporis 20 (M. canis 46, T. mentagrophytes 30). The most frequent localizations in adult were toenail and feet (63) (T. rubrum 47.3 and 45.7, T. mentagrophytes 9.5 and 23.4, respectively), fingernail 15.6 (C. albicans 25 and T. rubrum 8.3) and trunk and extremities 12.2 (M. furfur 57.5 and T. mentagrophytes 14.9). Direct examination allowed the diagnosis of 94.66 of all positive samples and 91.74 of the cases with positive isolation.(AU)

[Multicenter study of superficial mycoses in Argentina. EMMS Group]. / Estudio multicéntrico de micosis superficiales en Argentina. Grupo EMMS.

Dermatomycoses are an important sanitary problem. Our country counts with partial epidemiological data only referred to a few geographical areas. The aim of our work was to perform a multicenter study with the participation of 15 medical centers located at the Capital City and in 11 Provinces of Argentina with different socioeconomical and cultural characteristics. Between October and December 1998, 851 ambulatory patients of both sexes (58% female, 42% male) with lesions compatible with mycoses in skin, nails or scalp were studied. Only 51.6% of the patients had mycoses confirmed by direct microscopic examination and/ or culture. Dermatophytes caused 69.3% of the diagnosticated infections, yeast 12.2% and Malassezia furfur 13.3%. Agents of dermatomycoses most frequently isolated were T. rubrum (52.7%), T. mentagrophytes (19.2%) and M. canis (14.2%). C. albicans was the predominant causative agent of yeast infections (44.4%). In children below 11 years old, the most frequent infections were tinea capitis 55.4% (M. canis 60%, T. tonsurans and M. gypseum 8.3%) and tinea corporis 20% (M. canis 46%, T. mentagrophytes 30%). The most frequent localizations in adult were toenail and feet (63%) (T. rubrum 47.3% and 45.7%, T. mentagrophytes 9.5% and 23.4%, respectively), fingernail 15.6% (C. albicans 25% and T. rubrum 8.3%) and trunk and extremities 12.2% (M. furfur 57.5% and T. mentagrophytes 14.9%). Direct examination allowed the diagnosis of 94.66% of all positive samples and 91.74% of the cases with positive isolation.