Epidemiological investigation and typing of Candida glabrata clinical isolates by FTIR spectroscopy.

Candida glabrata has emerged as one of the leading agents of fungal infections and strain typing is essential for epidemiological investigation that is generally achieved by molecular techniques. In this work, we studied twenty-nine C. glabrata strains isolated from different patients, using a phenotypic approach based on Fourier Transform Infrared (FTIR) spectroscopy, which has been in a previous study successfully applied as a rapid typing method for Candida albicans. A two-step procedure was used for the analysis. The first step included sixteen strains for the internal validation phase, which aimed at finding the spectral windows that would provide the best differentiation between strains. In this phase, hierarchical cluster analysis (HCA) carried out using three spectral windows (900-1200, 1540-1800, 2800-3000 cm(-1)) allowed to obtain the best classification, where each patient strains could be clustered together. A genotypic technique based on randomly amplified polymorphic DNA-analysis (RAPD) confirmed these results. In a second step, the external validation phase, thirteen other clinical strains of C. glabrata isolated from multiple sites in four ICU patients, were tested by FTIR spectroscopy. The analysis was based on the spectral regions previously found in the first step. HCA classification of the strains gave four groups, one group per patient. These results suggest that no inter-human transmission took place. This study shows the potential of FTIR approach for typing of C. glabrata with several advantages compared to other techniques. FTIR typing is fast, effective, and reagent free. Moreover, it is applicable to all micro-organisms and requires a small quantity of biomass.

FTIR spectroscopy in medical mycology: applications to the differentiation and typing of Candida.

The incidence of fungal infections, in particular candidiasis and aspergillosis, has considerably increased during the last three decades. This is mainly due to advances in medical treatments and technologies. In high risk patients (e.g. in haematology or intensive care), the prognosis of invasive candidiasis is relatively poor. Therefore, a rapid and correct identification of the infectious agent is important for an efficient and prompt therapy. Most clinical laboratories rely on conventional identification methods that are based on morphological, physiological and nutritional characteristics. However, these have their limitations because they are time-consuming and not always very accurate. Moreover, molecular methods may be required to determine the genetic relationship between the infectious strains, for instance in Candida outbreaks. In addition, the latter methods require time, expensive consumables and highly trained staff to be performed adequately. In this study, we have applied the FTIR spectroscopic approach to different situations encountered in routine mycological diagnosis. We show the potentials of this phenotypic approach, used in parallel with routine identification methods, for the differentiation of 3 frequently encountered Candida species (C. albicans, C. glabrata and C. krusei) by using both suspensions and microcolonies. This approach, developed for an early discrimination, may help in the initial choice of antifungal treatment. Furthermore, we demonstrate the feasibility of the method for intraspecies comparison (typing) of 3 Candida species (C. albicans, C. glabrata and C. parapsilosis), particularly when an outbreak is suspected.

Identification and expression analysis of ABC protein-encoding genes in Toxoplasma gondii. Toxoplasma gondii ATP-binding cassette superfamily.

The ATP-binding cassette (ABC) transporters are one of the largest evolutionarily conserved families of proteins. They are characterized by the presence of nucleotide-binding domains (NBDs), which are highly conserved among organisms. In the present study, we used human and protozoan ABC sequences, and ATP-binding consensus motifs to screen the Toxoplasma gondii TwinScan2 predicted proteins database. We identified 24 ABC open reading frames (ORFs), whose deduced amino acid sequences exhibited all the typical biochemical features of the ABC family members. Fifteen of them clustered into five of the seven families of human ABC proteins: six ABCBs (drug, peptides and lipid export), two ABCCs (organic anion conjugates and drug export), one ABCE (Rnase L inhibitor, RLI, antibiotic resistance and translation regulation), one ABCF (drug resistance and regulation of gene expression) and five ABCGs (drug export and resistance). The nine other ORFs were represented by four ABCHs (energy-generating subunits), four SMCs (structural maintenance of chromosomes) and one member of unclear origin, whose closest homologue was the yeast Elf1 protein (mRNA export factor). A notable feature of the Toxoplasma ABC superfamily seems to be the absence of genes encoding ABCA and ABCD members. Expression analysis of ABC genes in tachyzoite and bradyzoite stages revealed the presence of ABC transcripts for all genes studied. Further research on the implication of these ABC proteins will increase our knowledge of the basic biology of Toxoplasma and provide the opportunity to identify novel therapeutic targets. To our knowledge, this is the first report of ABC transporters in T. gondii.

FTIR spectroscopy as a potential tool to analyse structural modifications during morphogenesis of Candida albicans.

Candida albicans is a polymorphic organism that grows under certain conditions as blastospores, hyphae or pseudohyphae. The potentials of FTIR spectroscopy for assessing structural differences in C. albicans blastospores and hyphae were investigated. The main observed differences were localised in the polysaccharide (950-1,185 cm(-1)), protein (1,480-1,720 cm(-1)), and the fatty acids (2,840-3,000 cm(-1)) regions. Quantitative evaluation of differences between hyphae and blastospores by curve-fitting of these regions indicate that these modifications could be due to both changes in structure and content of components of the cell wall such as beta-glucans, mannoproteins, and lipids. Furthermore, glycogen consumption could be involved during hyphae elongation. Thus, FTIR spectroscopy can be an interesting tool to investigate differences in structure and in content between blastospores and hyphae. We also demonstrate through this study that differentiation of C. albicans clinical strains using hyphae is feasible, as this has been previously shown with blastospores. This preliminary work on identification of C. albicans using hyphae is a prelude to a larger clinical study for early typing within 7 h from a pure culture.

Rapid identification of Candida species by FT-IR microspectroscopy.

Due to the continuous increase of human candidiasis and the great diversity of yeasts of the Candida genera, it is indispensable to identify this yeast as early as possible. Early identification enables an early diagnostic and patient-adapted anti-fungal therapy, thus reducing morbidity and mortality related to these infections. In view of this, we have in this study investigated microcolonies using a method based on Fourier transform-infrared microspectroscopy (FTIRM) for a rapid and early identification of the most frequent Candida species encountered in human pathology. FTIR spectroscopy is a whole-cell "fingerprinting" method by which microorganisms can be identified. By exploiting the huge discriminating capacity of this technique, we identified 6 species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, and Candida kefyr) from a collection of 57 clinical strains of Candida, isolated from hospitalised patients. Data obtained on 10- to 18-h-old microcolonies were compared to cultures of 24 h. Our results clearly show the efficiency and the robustness of FTIR (micro)spectroscopy in identifying species with a classification rate of 100% for both microcolonies and 24-h cultures. FTIR microspectroscopy is thus a promising clinical approach, because compared to conventional and molecular techniques, it is time and money saving, has great identification and discriminating potentials, and is amenable to an automated high-throughput routine system.

Evaluation of a strategy for Toxoplasma gondii oocyst detection in water.

Several recent outbreaks of toxoplasmosis were related to drinking water. We propose a strategy for Toxoplasma oocyst detection as part of an approach to detecting multiple waterborne parasites, including Giardia and Cryptosporidium spp., by the U.S. Environmental Protection Agency method with the same sample. Water samples are filtered to recover Toxoplasma oocysts and purified on a sucrose density gradient. Detection is based on PCR and mouse inoculation (bioassay) to determine the presence and infectivity of recovered oocysts. In an experimental seeding assay with 100 liters of deionized water, a parasite density of 1 oocyst/liter was successfully detected by PCR in 60% of cases and a density of 10 oocysts/liter was detected in 100% of cases. The sensitivity of the PCR assay varied from less than 10 to more than 1000 oocysts/liter, depending on the sample source. PCR was always more sensitive than mouse inoculation. This detection strategy was then applied to 139 environmental water samples collected over a 20-month period. Fifty-three samples contained PCR inhibitors, which were overcome in 39 cases by bovine serum albumin addition. Among 125 interpretable samples, we detected Toxoplasma DNA in 10 cases (8%). None of the samples were positive by mouse inoculation. This strategy efficiently detects Toxoplasma oocysts in water and may be suitable as a public health sentinel method.

P-glycoprotein inhibitors modulate accumulation and efflux of xenobiotics in extra and intracellular Toxoplasma gondii.

We examined xenobiotic transport and the effects of P-glycoprotein (Pgp) inhibitors on efflux function in Toxoplasma gondii tachyzoites. The fluorescence emission of JC-1 and daunorubicin (Pgp substrates) was determined in both extracellular tachyzoites and T. gondii-infected human KB cells. Dye accumulation and efflux were modulated by verapamil (Vp) and cyclosporin A (CsA), both of which are Pgp inhibitors. Red JC-1 emission was measured from 10(6) extracellular tachyzoites, using spectrofluorometry. The increase in red emission was significant from 1 microM concentration of both drugs and was higher with CsA than with Vp. Compared with untreated tachyzoites, JC-1 efflux was inhibited by 3 microM CsA and 3 microM Vp. With intracellular tachyzoites, the fluorescence distribution of daunorubicin (DNR) between the parasitophorous vacuole and the host cell was modulated by Vp and CsA. In media free of CsA and Vp, DNR emission inside intracellular tachyzoites was very weak, as observed by confocal microscopy. In the presence of CsA or Vp, DNR emission was markedly enhanced in tachyzoites but not in the whole vacuole. The modulation of DNR uptake seems to involve the tachyzoite membrane rather than the parasitophorous vacuole or host cell membranes. It suggests that Vp would inhibit the DNR efflux from intracellular tachyzoites through a transitory effect. In conclusion, these two Pgp inhibitors increase both extracellular and intracellular dye accumulation in living T. gondii, pointing to the existence of a transmembrane transport mediated by a Pgp homologue located on the parasite membrane complex.

Use of co-immunoelectrodiffusion to detect presumed disease-associated precipitating antibodies, and time-course value of specific isotypes in bird-breeder's disease.

BACKGROUND AND OBJECTIVE: The practical value of immunological diagnosis of bird-breeder's disease (BBD) is controversial, because of difficulties in distinguishing active disease patients from simple contact subjects. The aim of this study was to determine the diagnostic and prognostic value of (a) presumed disease-associated antibodies precipitating pigeon antigens (immunoglobulin A (IgAp) and P2 component), (b) characterization of specific isotypes (IgG, IgM, and IgA), and (c) antibody kinetics after antigen eradication. METHODS: 405 subjects (775 sera) in contact with birds were studied [by means of co-immunoelectrodiffusion (Co-IED) and enzyme-linked immunofiltration (ELIFA)] with soluble extracts of pigeon droppings and squab crop milk. These patients were divided into two groups based on the final clinical evaluation of the patients' physicians, which was taken as the gold standard (positive in 90 and negative in 315 cases). RESULTS: On the basis of this gold standard, the detection of presumed disease-associated precipitating antibodies by Co-IED had a specificity of 95.5%, a sensitivity of 98.7%, an accuracy of 98%, and positive and negative predictive values of 95.5% and 98.7%, respectively. Most of the patients with a final positive diagnosis of BBD had specific IgG, IgM, and IgA antibodies by ELIFA. After antigen eradication, anti IgAp and/or P2 antibodies disappeared more rapidly than other precipitating systems. CONCLUSION: Identification by Co-IED of precipitating immune complexes IgAp and/or P2 significantly reinforces the intrinsic credibility of immunological diagnosis of BBD. Compared to these presumed disease-associated precipitating antibodies, detection and time course of specific IgM, IgA antibodies, provided no additional diagnostic value or prognostic arguments to judge disease activity after antigen eradication.

Virulencia en Toxoplasma gondii: ¿Las presentaciones clínicas inusuales en la Amazonía pueden poner en evidencia factores de virulencia? / Virulence of Toxoplasma gondii: ¿Uncommon clinical presentation as evidence on virulence factors?

Toxoplasma gondii es el agente responsable de la toxoplasmosis, una de las infecciones parasitarias más frecuentes en el mundo. Toxoplasma gondii pertenece al phylum Apicomplexa o Sporozoa, la clase de los Coccidea y a la orden de los Eimeriida. La infección en el hombre es habitualmente asintomática o puede tener un curso clínico benigno. Habitualmente solo 15-30/100 de los pacientes inmunocompetentes presentan algún síntoma que consiste generalmente en un síndrome ganglionar que se autolimita. En raras ocasiones una toxoplasmosis adquirida en la edad adulta pude dar lugar a una retinocoroiditis. La frecuencia de esto no ha sido determinada por estudios longitudinales amplios pero en un brote en Atlanta (USA) solo 1 de 37 individuos desarrolló la enfermedad ocular luego de 4 años de seguimeinto. En un brote bien documentado en Nueva York, uno de siete miembros de una familia desarrolló coriorretinitis por Toxoplasma 129 días luego de la infección y en un brote reciente en Vancouver el número de pacientes sintomáticos oculares fue 19 de un total de 100 sintomáticos. En Colombia la toxoplasmosis congénita es aún un problema de salud pública importante. Según estudios realizados en diferentes regiones, cada año aparecen 2 a 10 por cada 1.000 recién nacidos con toxoplasmosis congénita. Actualmente, el Ministerio de Salud no tiene un programa de control para la enfermedad.

Frequency of specific anti-Toxoplasma gondii IgM, IgA and IgE in Colombian patients with acute and chronic ocular toxoplasmosis

We studied the frequency of specific anti-Toxoplasma IgM, IgA and IgE antibodies in serum of 28 immunocompetent Colombian patients, selected by ophthalmologists and with lesions that were compatible with ocular toxoplasmosis. Patients were classified in three groups: (i) group 1 consisted of ten patients with a first episode; (ii) group 2, with seven patients with a recurrence and (iii) group 3, consisted of eleven patients with chronic chorioretinal lesion without uveitis. We found that 10/28 (35 per cent) of Colombian patients with ocular toxoplasmosis possessed at least one serological marker for Toxoplasma infection different from IgG. In group 1 (first episode), we found simultaneous presence of specific IgM plus IgA plus IgE in 1/10 (10 per cent). In group 2 (recurrences) in 1/7 (14 per cent) we found IgM and IgA test positives and in 1/7 (14 per cent) we found IgM and IgE tests positives. In group 3 (toxoplasmic chorioretinal scar) the IgA serological test was positive in 2/11 (18 per cent). These results show that serum IgM or IgA or IgE can be present during recurrences.

Mecanismos efectores del interferón gamma contra la infección por Toxoplasma gondii / Effector mechanisms of gamma interferon against Toxoplasma gondii

Toxoplasma gondii es un protozoario parásito de desarrollo intracelular. La enfermedad humana producida por T. gondii es una causa importante de morbilidad y mortalidad neonatal y en pacientes inmunodeprimidos. En esta reseña, se revisan los conocimientos actuales sobre los mecanismos efectores del interferón gamma (IFN gamma), la principal citocina protectora contra T. gondii, incluyendo los resultados que hemos obtenido en un modelo de infección in vitro de células humanas monocitarias (THP1). El IFN gamma protege las células humanas contra T. gondii por mecanismos diferentes a aquéllos con los que protege las células de ratón. En el modelo de infección de células THP1, el IFN gamma protege por dos mecanismos independientes. Un primer efecto es la reducción del porcentaje de células parasitadas, el cual está ligado a una disminución de la actividad PLA subíndice 2 parasitaria y celular. Se trata de un nuevo mecanismo efector del IFN gamma contra la infección toxoplásmica, diferente a los mecanismos parasiticidas y parasitostáticos previamente descritos. Un segundo efecto ocurre por interrupción del crecimiento parasitario intracelular a través de la activación de la enzima idoleamina-oxigenasa que degrada el triptófano, lo que constituye un efecto parasitostático. Contrariamente a lo que ocurre en el modelo de infección de células monocitarias de ratón, la producción de óxido nítrico (NO) no juega un papel determinante en los monocitos humanos

Avances diagnósticos en toxoplasmosis. PCR, nuevos marcadores de infección evolutiva y otras técnicas

El diagnóstico de la toxoplasmosis requiere la realización de pruebas de laboratorio; en esta revisión se presenta un sumario de las técnicas que han aparecido en los últimos años y que han aportado nuevos criterios para afirmar la presencia de una infección clínicamente importante. Se discute la cinética de anticuerpos y los límites y ventajas de las técnicas de detección de anticuerpos y del parásito, incluyendo la reacción en cadena de la polimerasa (PCR). Se presenta una guía para la interpretación de resultados en las diversas manifestaciones clínicas de esta infección. Grandes avances y mejorías notables en las técnicas de diagnóstico se han realizado se hace aún necesario mejorarlas y simplificarlas para lograr un mayor acceso a su aplicación.