[Show abstract][Hide abstract] ABSTRACT: Giardia duodenalis (syn. lamblia; syn. intestinalis) susceptibility testing is not routinely performed because the classical culture methods are very time-consuming and laborious. We developed a novel flow cytometry (FC) assay to evaluate the susceptibility of G. duodenalis trophozoites to metronidazole (MTZ). Different concentrations of MTZ were added to cultures of trophozoites (105/mL) and the cultures were incubated for different periods. The 50% inhibitory concentration was calculated and propidium iodide (PI) was used to quantify the number of dead cells. After treatment, PI-positive trophozoites increased with increasing drug concentration and exposure time. An excellent correlation was found between FC and the classical method. A novel, accurate and reliable method is now available to evaluate G. duodenalis viability.
Memórias do Instituto Oswaldo Cruz 11/2014; · 1.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Candida krusei is an important agent of opportunistic infections, often displaying resistance to several antifungals. We describe the in vivo acquisition of resistance to voriconazole (VRC) by C krusei isolates recovered from a leukemia patient under a long period of VRC therapy. In order to mimic the in vivo development of VRC resistance, a susceptible C. krusei isolate was daily exposed to VRC 1μg/ml in vitro. Interestingly, after 5 days of exposure to VRC, a MIC of 4 μg/ml was achieved; this value remaining constant after 25 additional days of daily incubation with VRC, and also after 30 consecutive days of daily incubation in VRC-free medium. Our objective was to unveil the associated molecular resistance mechanisms, such as expression of efflux pumps genes and ERG11 gene mutations, among the resistant strains. A synergistic effect was found between the efflux blocker Tacrolimus (FK506) and VRC in all the resistant strains. Moreover, ABC1 gene expression increased over time in both the in vivo and in vitro induced resistant strains, contrarily to ABC2 and ERG11 genes, whose expression was invariably lower and constant. ERG11 gene sequencing showed two different types of mutations: heterozygosity in C. krusei strains at T1389T/C, corresponding to a synonymous mutation, and a missense mutation, at position T418C, resulting in a change from Tyr to His, among resistant C. krusei clinical isolates. This study highlights the relevance of ATP-dependent efflux pumps activity, namely Abc1p, in VRC antifungal resistance and describes new mutations in ERG11 gene among resistant C. krusei clinical isolates.
Antimicrobial Agents and Chemotherapy 05/2014; · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Malassezia yeasts have long been considered commensal fungi, unable to elicit significant damage. However, they have been associated with a diversity of cutaneous diseases, namely pityriasis versicolor, Malassezia folliculitis, seborrheic dermatitis, atopic dermatitis, psoriasis, and confluent and reticulate papillomatosis. Several hypotheses have been proposed to explain the pathogenic mechanisms of these fungi, but none have been confirmed. More recently, such organisms have been increasingly isolated from bloodstream infections raising serious concern about these fungi. Given the difficulty to culture these yeasts to proceed with speciation and antimicrobial susceptibility tests, such procedures are most often not performed and the cutaneous infections are treated empirically. The recurring nature of superficial skin infections and the potential threat of systemic infections raise the need of faster and more sensitive techniques to achieve isolation, identification, and antimicrobial susceptibility profile. This article reviews and discusses the latest available data concerning Malassezia infections and recent developments about diagnostic methods, virulence mechanisms, and susceptibility testing.
Journal of the American Academy of Dermatology 02/2014; · 5.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cryptosporidium spp., Giardia lamblia y Encephalitozoon intestinalis, oportunistas emergentes Cryptosporidium spp., Giardia lamblia y Encephalitozoon intestinalis emergent opportunists RESUMEN Es un hecho conocido que las infecciones oportunistas por protozoos y hongos han aumentado en los últimos años, debido especialmente al aumento de las infecciones por VIH. Cryptosporidium spp., Giardia lamblia y Encephalitozoon intestinalis son protozoos y hongo, respectivamente, mundialmente reconocidos como agentes oportunistas emergentes, responsables de brotes epidémicos provocados por la ingestión de agua potable contaminada, incluso después de una correcta desinfección. La ingestión de estos protozoos puede provocar diferentes grados de enfermedad, entre aguda o leve (población sana) hasta situaciones más graves y agresivas, hasta a veces mortales (pacientes inmunocomprometidos y/o inmunodeprimidos). A pesar de ser responsables de muchos brotes epidémicos, su diagnóstico de laboratorio permanece arduo y trabajoso, incluso utilizando las nuevas técnicas desarrolladas en los últimos años. En esta revisión se resumen las consideraciones generales de estos oportunistas emergentes, así como los métodos de diagnóstico más usuales, incluso los más recientes y específicos. Palabras clave: Oportunistas emergentes, protozoos, hongos, Cryptosporidium spp., Giardia lamblia, Encephalitozoon intestinalis. SUMMARY Epidemiological data, regarding parasitic and fungi opportunist infections, have changed in the last years, especially due to HIV infection. Cryptosporidium spp., Giardia lamblia and Encephalitozoon intestinalis are protozoan and fungi, respectively, worldwide known as opportunistic emergent agents, being responsible by epidemic outbreaks after ingestion of contaminated water, even following a correct disinfection treatment. Its ingestion can cause different effects on individuals' health, from light or acute among the healthy population, to serious, aggressive or even deadly among the immunodepressed or immunocompromised patients. Contaminated water ingestion can result in outbreaks but protozoa laboratory diagnosis still remains very laborious, even after the development of more sensitive and specific techniques in the last years. In this paper, a revision of these emergent opportunists, their main characteristics and diagnostic tools are described, including the most recent and specific techniques.
[Show abstract][Hide abstract] ABSTRACT: Pneumocystis jirovecii is an opportunistic pathogen responsible for severe pneumonia in immunocompromised patients. Flow cytometry (FC) is a method widely used in different areas of clinical diagnosis like hematology and immunology. Recently it has started to be used in microbiology with a great potential for diagnosis of emergent microorganisms in clinical samples, especially when present in low numbers. The detection of Pneumocystis jirovecii in respiratory samples can be performed by FC, using specific monoclonal antibodies. Considering clinical diagnosis as a reference method, we previously showed FC to be 100% sensitive and specific when compared to immunofluorescence. Being an automated method, it is faster and less subject to human error than microscopic evaluation.
[Show abstract][Hide abstract] ABSTRACT: The mechanism(s) responsible for breast capsular contracture (CC) remain unknown, but inflammatory pathways play a role. Various molecules have been attached to implant shells in the hope of modifying or preventing CC. The intrinsic antibacterial and antifungal activities of chitosan and related oligochitosan molecules lend themselves well to the study of the infectious hypothesis; chitosan's ability to bind to growth factors, its hemostatic action, and its ability to activate macrophages, cause cytokine stimulation, and increase the production of transforming growth factor (TGF)-β1 allow study of the hypertrophic scar hypothesis.
The authors perform a comprehensive evaluation, in a rabbit model, of the relationship between CC and histological, microbiological, and immunological characteristics in the presence of a chitooligosaccharide (COS) mixture and a low molecular weight chitosan (LMWC).
Eleven adult New Zealand rabbits were each implanted with three silicone implants: a control implant, one impregnated with COS, and one impregnated with LMWC. At four-week sacrifice, microdialysates were obtained in the capsule-implant interfaces for tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8) level assessment. Histological and microbiological analyses were performed.
Baker grade III/IV contractures were observed in the LMWC group, with thick capsules, dense connective tissue, and decreased IL-8 levels (p < .05) compared to control and COS groups. Capsule tissue bacterial types and microdialysate TNF-α levels were similar among all groups.
Chitosan-associated silicone implantation in a rabbit model resulted in Baker grade III/IV CC. This preclinical study may provide a model to test various mechanistic hypotheses of breast capsule formation and subsequent CC.
Aesthetic Surgery Journal 07/2011; 31(5):540-50. · 2.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A prospective study to assess fungaemia was conducted for 12 months at a Portuguese University Hospital. A total of 35 Candida albicans isolates obtained from 12 patients with fungaemia were compared by a multiplex PCR system using four microsatellite loci. Blood isolates were evaluated against concomitant isolates from urine, lower respiratory secretions and central venous catheters, as well as with successive isolates recovered from recurrent episodes of fungaemia. The data analyzed included the department of admission, underlying diseases and antifungal therapy. The susceptibility phenotypes of all isolates to amphotericin B, fluconazole, itraconazole, voriconazole and caspofungin were determined according to the CLSI M27-A3 protocol. We observed a high degree of similarity between successive blood isolates and between blood and concomitant isolates from other sites of the same patient. This is suggestive of the recurrence of fungaemia and was due to the same strain, possibly as a result of the failure of antifungal therapy. The genetic similarity observed between some strains isolated from different patients suggested the likelihood that they were hospital acquired. Distinct patients were infected by the same strain at different time periods, and an increase in antifungal resistance was observed over time for some of these strains. Hospital-acquired exogenous nosocomial infections can be associated with higher risks of antifungal resistance and need to be closely monitored. Particular attention should also be given to endogenous non-blood Candida isolates which can be critical in high risk patients, as they often can become invasive in immunodeficient individuals.
Medical mycology: official publication of the International Society for Human and Animal Mycology 04/2011; 49(3):248-52. · 2.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The VITEK 2 automated system (bioMérieux) is one of the most widely used instruments in clinical microbiology laboratories for the identification and evaluation of the susceptibility profiles of bacteria including the detection of extended-spectrum β-lactamases (ESBLs) produced by Escherichia coli, Klebsiella pneumoniae and Klebsiella oxytoca. Currently, the Clinical and Laboratory Standards Institute recommends the use of ESBL confirmatory tests in addition to standard susceptibility testing. In order to evaluate the accuracy of VITEK 2-positive results regarding clinical isolates of E. coli (n = 110) and K. pneumoniae (n = 72), four additional ESBL detection systems were compared: the Phoenix Automated Microbiology System (BD Diagnostic Systems) and the MicroScan WalkAway-96 System (Dade Behring), and two manual systems as confirmatory tests, the Etest (AB Biodisk) and double disc diffusion (DDS) test. Epidemiological data regarding the tested strains were also collected and their susceptibility phenotypes were determined. The four methods resulted in concordant results for 126 of the 182 strains. However, the different tests displayed distinct results: the VITEK 2 system was in disagreement in 23.9 % of cases with DDS, in 15.3 % with Etest, in 23 % with the MicroScan WalkAway-96 System and in 23.6 % with the Phoenix Automated Microbiology System. Epidemiological data indicated that the majority of ESBL-positive E. coli strains were isolated from patients admitted to internal medicine wards (72.7 %), whilst K. pneumoniae ESBL-positive isolates were equally distributed between internal medicine wards (45.8 %) and intensive care units (45.8 %). Most of these strains were isolated from urine. In contrast to ESBL-negative isolates, the ESBL-positive strains displayed multiple drug resistance, namely to quinolones, aminoglycosides and trimethoprim-sulfamethoxazole. No significant resistance to carbapenems was detected. Overall, this study demonstrates the need for a confirmatory test following positive ESBL detection with the VITEK 2 system (panel AST-037), which appears to yield a large number of false-positive results.
Journal of Medical Microbiology 02/2011; 60(Pt 6):756-60. · 2.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This is the first case report of Candida glabrata-disseminated candidiasis describing the acquisition of echinocandin resistance following anidulafungin treatment. The initial isolates recovered were susceptible to echinocandins. However, during 27 days of anidulafungin treatment, two resistant strains were isolated (from the blood and peritoneal fluid). The resistant peritoneal fluid isolate exhibited a Ser663Pro mutation in position 1987 of FKS2 HS1 (hot spot 1), whereas the resistant blood isolate displayed a phenylalanine deletion (Phe659).
Antimicrobial Agents and Chemotherapy 12/2010; 55(3):1312-4. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Flow cytometry has found wide applications in areas like haematology and immunology, but also presents great potential in microbiology. The susceptibility of clinical isolates of Candida and Cryptococcus to antifungal compounds can be assayed by flow cytometry using fluorescent probes like FUN-1, propidium iodide and JC-1, with several advantages. Following 1 or 2 h of incubation, depending on the antifungal compound, versus 48 h of the classical methods, it is possible to establish the different susceptibility profiles. Additionally, it provides information regarding the mechanisms of action and might infer about resistance mechanisms.
[Show abstract][Hide abstract] ABSTRACT: The diagnosis of candida balanitis should be based upon both clinical and mycological data. The procedure of material collection is a critical issue to confirm or rule out the clinical diagnosis of candida balanitis.
To compare direct impression of the glans on the agar surface of solid culture media with the collection of genital exudates with cotton swab for the diagnosis of candida balanitis.
A prospective cross-sectional study was carried out during a 36-month period. Sexually transmitted disease clinic attendees with balanitis and asymptomatic men were included. Specimens for yeast culture were collected from the glans penis and inner preputial layer using the direct impression on CHROMagar candida medium and by swabbing with a sterile cotton swab.
Among 478 men enrolled, 189 had balanitis. The prevalence of candida balanitis was 17.8% (85/478) confirmed after culture by direct impression; the swab method detected only 54/85 (63.5%) of these men. Of the 289 asymptomatic men, 36 (12.5%) yielded Candida spp; the swab method detected only 38.9% of these men. The risk of having candida balanitis is 8.9 (IC 95% 2.48 to 32.04) whenever the number of candida colonies recovered by direct impression was greater than 10.
Direct impression on CHROMagar candida medium resulted in the highest Candida spp recovery rate. More than 10 colonies yielded by impression culture were statistically associated with candida balanitis. This method shows the ideal profile for sampling the male genital area for yeasts and should be included in the management of balanitis.
[Show abstract][Hide abstract] ABSTRACT: Encephalitozoon intestinalis is responsible for intestinal disease in patients with AIDS and immunocompetent patients. The infectious form is a small spore that is resistant to water treatment procedures. Its detection is very important, but detection is very cumbersome and time-consuming. Our main objective was to develop and optimize a specific flow cytometric (FC) protocol for the detection of E. intestinalis in hospital tap water and human feces. To determine the optimal specific antibody (Microspor-FA) concentration, a known concentration of E. intestinalis spores (Waterborne, Inc.) was suspended in hospital tap water and stool specimens with different concentrations of Microspor-FA, and the tap water and stool specimens were incubated under different conditions. The sensitivity limit and specificity were also evaluated. To study spore infectivity, double staining with propidium iodide (PI) and Microspor-FA was undertaken. Distinct approaches for filtration and centrifugation of the stool specimens were used. E. intestinalis spores stained with 10 microg/ml of Microspor-FA at 25 degrees C overnight provided the best results. The detection limit was 5 x 10(4) spores/ml, and good specificity was demonstrated. Simultaneous staining with Microspor-FA and PI ensured that the E. intestinalis spores were dead and therefore noninfectious. With the stool specimens, better spore recovery was observed with a saturated solution of NaCl and centrifugation at 1,500 x g for 15 min. A new approach for the detection of E. intestinalis from tap water or human feces that ensures that the spores are not viable is now available and represents an important step for the prevention of this threat to public health.
[Show abstract][Hide abstract] ABSTRACT: Candida parapsilosis is a common isolate from clinical fungal infectious episodes. Resistance of C. parapsilosis to azoles has been increasingly reported. To analyse the development of resistance in C. parapsilosis, four azole-susceptible clinical strains and one American Type Culture Collection type strain were cultured in the presence of fluconazole, voriconazole and posaconazole at different concentrations. The isolates developed variable degrees of azole resistance according to the antifungal used. Fluconazole was the fastest inducer while posaconazole was the slowest. Fluconazole and voriconazole induced resistance to themselves and each other, but not to posaconazole. Posaconazole induced resistance to all azoles. Developed resistance was stable; it could be confirmed after 30 days of subculture in drug-free medium. Azole-resistant isolates revealed a homogeneous population structure; the role of azole transporter efflux pumps was minor after evaluation by microdilution and cytometric assays with efflux pump blockers (verapamil, ibuprofen and carbonyl cyanide 3-chloro-phenylhydrazone). We conclude that the rapid development of azole resistance occurs by a mechanism that might involve mutation of genes responsible for ergosterol biosynthesis pathway, stressed by exposure to antifungals.
FEMS Yeast Research 05/2009; 9(4):626-33. · 2.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several mechanisms may be associated with Candida albicans resistance to azoles. Ibuprofen was described as being able to revert resistance related to efflux activity in Candida. The aim of this study was to uncover the molecular base of antifungal resistance in C. albicans clinical strains that could be reverted by ibuprofen. Sixty-two clinical isolates and five control strains of C. albicans were studied: the azole susceptibility phenotype was determined according to the Clinical Laboratory for Standards Institute, M27-A2 protocol and minimal inhibitory concentration values were recalculated with ibuprofen (100 microg mL(-1)); synergistic studies between fluconazole and FK506, a Cdr1p inhibitor, were performed using an agar disk diffusion assay and were compared with ibuprofen results. Gene expression was quantified by real-time PCR, with and without ibuprofen, regarding CDR1, CDR2, MDR1, encoding for efflux pumps, and ERG11, encoding for azole target protein. A correlation between susceptibility phenotype and resistance gene expression profiles was determined. Ibuprofen and FK506 showed a clear synergistic effect when combined with fluconazole. Resistant isolates reverting to susceptible after incubation with ibuprofen showed CDR1 and CDR2 overexpression especially of the latter. Conversely, strains that did not revert displayed a remarkable increase in ERG11 expression along with CDR genes. Ibuprofen did not alter resistance gene expression significantly (P>0.05), probably acting as a Cdrp blocker.
FEMS Yeast Research 03/2009; 9(4):618-25. · 2.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Balanitis is defined as inflammation of the glans penis, often involving the prepuce (balanoposthitis). It is a common condition due to a wide variety of causes with infection being the most frequent and several microorganisms reported. The clinical aspect is often non specific. The management of balanoposthitis remains a clinical challenge.
To evaluate the prevalence of infectious balanitis, its management, clinical features, laboratory procedures and treatment options.
One hundred eighteen patients with infectious balanitis were evaluated between 1995 and 2004 and laboratory data were collected.
Balanitis was diagnosed in 219 (10.7%) of the men that have attended the sexually transmitted disease (STD) Clinic. One hundred eighteen (53.9%) had clinically been assumed to suffer from infectious balanitis. In 75 (63.6%) patients the diagnosis was confirmed by culture studies. Candida albicans was isolated from 24 patients. Staphylococcus spp. and groups B and D Streptococci were the most frequently isolated bacteria. All men were uncircumcised. Ninety-one (77.1%) of infectious balanitis patients were treated with antifungal agents. Twelve patients with infectious noncandida balanitis were treated with general antibiotic therapy. Fifty-five (46.6%) patients had a follow-up of 3 to 12 months during which recurrences were registered in 7 (12.7%) patients.
Infectious balanitis was a common condition, affecting 53.9 % of male STD clinic patients in this study. Candida spp. were the most frequently isolated microorganisms. The clinical aspect is of little value in predicting the infectious agent associated with balanoposthitis.
International journal of dermatology 03/2009; 48(2):121-4. · 1.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Giardia lamblia is responsible for causing diarrhoeal diseases in humans. Infection occurs by fecal-oral route and is considered an important water pathogen, since many water surfaces are infected by cysts. Most studies involve cyst concentration procedures, followed by conventional microscopy, but are often tedious and influenced by fatigue. Our main objective was to optimize a specific flow cytometric (FC) protocol for detection of G. lamblia, to establish its sensibility limit and also the cyst viability. G. lamblia cysts (Waterborne, Inc., USA) were used for protocol optimization. FC analysis was performed using cyst suspensions stained with serial concentrations of a fluorescein-labelled mouse monoclonal antibody (Giardia-a-Glo, Waterborne). Serial concentrations (2 x 10(5), 1 x 10(5), 2 x 10(4), 1 x 10(4), 2 x 10(3), 1 x 10(3), 2 x 10(2) and 1 x 10(2)cysts/ml) were stained with the optimized antibody concentration and analysed by FC. Specificity and sensibility limit of the method were established using both prokaryotic (Escherichia coli, Staphylococcus aureus) and eukaryotic microorganisms (Candida albicans, Cryptosporidium parvum oocysts). Dead cysts were stained with 5.0 microg/ml of propidium iodide (PI, Sigma), with and without the specific fluorescent antibody. As the antibody concentration decreased, a decline of peak intensity was registered; 1.5 microg/ml was considered as the optimal antibody concentration, yielding a histogram clearly separated. We established a threshold of detection of 2 x 10(2)cysts/ml. Below threshold limit fluorescence was not enough to allow the discrimination of cysts. The staining procedure was shown to be specific, no cross-reaction occurring with bacteria, fungi or parasites. When using both antibody and PI, we could distinguish the viable cyst. With the use of specific antibodies, a distinct cellular population corresponding to cysts could be represented in the FC histogram.
Travel Medicine and Infectious Disease 08/2008; 6(4):234-9. · 1.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Haemato-Oncology Unit, Hospital S. Joao, suffered extensive refurbishing intervention in order to adapt for autotransplant patients. Eight new individual rooms with central HEPA filtration system were built. All patients admitted in the department during 14 months prior to and 14 months after renovation works were enrolled. A total of 403 admissions were considered and a detailed analysis of all patients with fungal infections, air quality and antifungal consumption were evaluated in order to study clinical, environmental and economical impact after unit renovation.
Patients with acute myeloid leukaemia submitted to induction treatment were the most susceptible to acquisition of fungal infections. Fungal infections were reduced after installation of HEPA filters in individual rooms, particularly proven and probable fungal infections. No patients were diagnosed with proven or probable mould infection in the period after the unit renovation and no deaths were registered among patients with the diagnosis of possible fungal infection. Considering the group of patients diagnosed with fungal infection, the average of hospitalization was reduced 3 d in the latter period. The new high-protected rooms showed a reduction of 50% and 95% of airborne fungi, respectively in the first week and after the second week. The consumption of voriconazole and caspofungin was reduced, respectively, 66% and 59% and the final cost with antifungal therapy was reduced by 17.4%.
Autotransplant patients may be under higher risk of infection, however, the installation of high-protective measures may efficiently prevent fungal infections in these patients. Renovation of haematology unit resulted in major clinical, environmental and economical improvements. The definition of reference values for airborne agents in hospital facilities remains urgent.
European Journal Of Haematology 06/2008; 80(5):436-43. · 2.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High-efficiency particulate air (HEPA) filters do not completely prevent nosocomial fungal infections. The first aim of this study was to evaluate the impact of different filters and access conditions upon airborne fungi in hospital facilities. Additionally, this study identified fungal indicators of indoor air concentrations.
Eighteen rooms and wards equipped with different air filter systems, and access conditions were sampled weekly, during 16 weeks. Tap water samples were simultaneously collected.
The overall mean concentration of atmospheric fungi for all wards was 100 colony forming units/m(3). We found a direct proportionality between the levels of the different fungi in the studied atmospheres. Wards with HEPA filters at positive air flow yielded lower fungal levels. Also, the existence of an anteroom and the use of protective clothes were associated to the lowest fungal levels. Principal component analysis showed that penicillia afforded the best separation between wards' air fungal levels. Fungal strains were rarely recovered from tap water samples.
In addition to air filtration systems, some access conditions to hospital units, like presence of anteroom and use of protective clothes, may prevent high fungal air load. Penicillia can be used as a general indicator of indoor air fungal levels at Hospital S. João.
American journal of infection control 04/2008; 36(2):129-34. · 3.01 Impact Factor