RESUMO
We evaluated the usefulness of a serum Aspergillus PCR assay for the diagnosis and prognosis of invasive aspergillosis in a study involving 941 patients for a total of 5146 serum samples. Fifty-one patients had proven/probable aspergillosis. We compared galactomannan (GM), PCR and mycologic analysis of pulmonary samples in both neutropenic and nonneutropenic patients. PCR performed in serum yielded 66.7% sensitivity, 98.7% specificity, 75.6% positive predictive value and 98.0% negative predictive value, while the GM index yielded 78.4% sensitivity, 87.5% specificity, 27% positive predictive value and 98.6% negative predictive value. The inclusion of PCR in the European Organization for Research and Treatment of Cancer (EORTC) and the Mycosis Study Group (MSG) mycologic criteria permitted the reclassification of nine other cases from possible to probable aspergillosis and increased the sensitivity to 71.7%. Combining the GM index with serum PCR increased the detection rate of invasive aspergillosis with 88.2% sensitivity. PCR was systematically negative in 16 patients with noninvasive forms of aspergillosis (namely aspergilloma and chronic aspergillosis). Remaining PCR positive after a period of 14 to 20 days of treatment was related to poor outcome at 30 and 90 days. Our results also indicate that, unlike the determination of the GM index, the initial fungus load as determined by PCR was highly predictive of 90-day mortality, with the rate of the latter being 15.8% for patients with <150 copies/mL vs. 73.2% for patients at or above that cutoff (p <0.0001). Therefore, PCR appears to be a powerful and interesting tool for the identification of patients with invasive aspergillosis who might benefit from more intense care.
Assuntos
Aspergillus/isolamento & purificação , Aspergilose Pulmonar Invasiva/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase/métodos , Soro/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Aspergillus/genética , Feminino , Galactose/análogos & derivados , Humanos , Masculino , Mananas/sangue , Pessoa de Meia-Idade , Neoplasias/complicações , Neutropenia/complicações , Valor Preditivo dos Testes , Prognóstico , Estudos Retrospectivos , Sensibilidade e Especificidade , Adulto JovemRESUMO
Candida inconspicua and Candida (Pichia) norvegensis are two emerging pathogenic species that exhibit reduced susceptibility to azole derivatives. Conventional (biochemical) approaches do not readily differentiate between the two species. The first aim of this work was to analyze the performance of biochemical, proteomic (matrix-assisted laser desorption ionization-time of flight [MALDI-TOF]), and molecular approaches in the precise identification of these species. These results then led us to sequence 3 genomic loci, i.e., the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA), the D1/D2 domain of the 28S rDNA, and the elongation factor 1α (EF-1α) gene, either directly or following cloning, of 13 clinical isolates and 9 reference strains belonging to the 5 species included in the Pichia cactophila clade, namely, Pichia cactophila, Pichia insulana, C. inconspicua, C. norvegensis, and P. pseudocactophila. Finally, isolates of C. inconspicua were challenged for sexual reproduction on the appropriate medium. Our results show that EF-1α sequencing and proteic profiling by MALDI-TOF are the two most efficient approaches to distinguish between C. norvegensis and C. inconspicua. As a characteristic of the P. cactophila clade, we found multiple alleles of the rDNA regions in certain strains belonging to the tested species, making ITS or D1/D2 sequencing not appropriate for identification. Whatever the method of identification, including MALDI-TOF and EF-1α sequencing, none could differentiate C. inconspicua from P. cactophila. The results of phylogenetic analysis and the generation of asci from pure cultures of all C. inconspicua strains both support the identification of P. cactophila as the teleomorph of C. inconspicua.
Assuntos
Candida/classificação , Cruzamentos Genéticos , Ordem dos Genes , Candida/química , Candida/genética , Candida/metabolismo , Candidíase/microbiologia , Análise por Conglomerados , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Humanos , Dados de Sequência Molecular , Técnicas de Tipagem Micológica , Fator 1 de Elongação de Peptídeos/genética , Filogenia , Proteoma/análise , RNA Ribossômico 28S/genética , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Hepatitis B (HBV) virus infection is characterized by the overproduction of subviral particles (SVP) over infectious Dane particles (VP). Precise regulation of the ratio between these forms is unknown, but its fluctuation may have a clinical impact. An enrichment method was applied to assess the SVP/VP ratio in chronically infected patients (CHB) and to compare the sensitivity of HBs antigen (HBsAg) and DNA detection methods. Plasmas from 9 genotype A-D CHB patients were fractionated on Nycodenz(®) gradients, and both HBV DNA and HBsAg were quantified in each collected fraction using standardized techniques expressed in IU/mL. Infection of primary human hepatocytes (PHHs) was performed with crude or fractionated plasma. Independently of the genotype, all plasmas showed a similar rate-zonal separation profile characterized by a bottom DNA-enriched peak surmounted by HBsAg-enriched fractions. Inoculation of PHH with plasma-derived VP-enriched fractions led to long-lasting production of virus in cell supernatants with a SVP/VP ratio similar to that observed in patient plasmas. In the VP fraction, one IU of HBsAg corresponded to approximately 5 million IU of HBV DNA. Rate-zonal gradient separation directly applied on patient plasma allows a better insight into the distribution of VP in HBeAg-positive CHB carriers. This study highlights the sensitivity difference of the techniques classically used to monitor HBV infection and indicates that VP-associated HBsAg contributes modestly to the overall amount of total circulating HBsAg in CHB. Such a fractionation approach should help to understand the fine regulation of HBsAg production over replication at different stages of CHB.
Assuntos
DNA Viral/sangue , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/virologia , Adulto , Células Cultivadas , Testes Diagnósticos de Rotina/métodos , Hepatócitos/virologia , Humanos , Sensibilidade e EspecificidadeRESUMO
PCR detection of Toxoplasma gondii in blood has been suggested as a possibly efficient method for the diagnosis of ocular toxoplasmosis (OT) and furthermore for genotyping the strain involved in the disease. To assess this hypothesis, we performed PCR with 121 peripheral blood samples from 104 patients showing clinical and/or biological evidence of ocular toxoplasmosis and from 284 (258 patients) controls. We tested 2 different extraction protocols, using either 200 µl (small volume) or 2 ml (large volume) of whole blood. Sensitivity was poor, i.e., 4.1% and 25% for the small- and large-volume extractions, respectively. In comparison, PCR with ocular samples yielded 35.9% sensitivity, while immunoblotting and calculation of the Goldmann-Witmer coefficient yielded 47.6% and 72.3% sensitivities, respectively. Performing these three methods together provided 89.4% sensitivity. Whatever the origin of the sample (ocular or blood), PCR provided higher sensitivity for immunocompromised patients than for their immunocompetent counterparts. Consequently, PCR detection of Toxoplasma gondii in blood samples cannot currently be considered a sufficient tool for the diagnosis of OT, and ocular sampling remains necessary for the biological diagnosis of OT.
Assuntos
Sangue/parasitologia , DNA de Protozoário/isolamento & purificação , Olho/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase/métodos , Toxoplasma/isolamento & purificação , Toxoplasmose Ocular/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA de Protozoário/genética , Feminino , Humanos , Immunoblotting/métodos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Toxoplasma/genética , Adulto JovemRESUMO
Invasive infections caused by filamentous fungi are a major threat for immunocompromised patients. Innate/acquired resistance to antifungal drugs might necessitate combination therapies. We assessed the potential combination of voriconazole with miltefosine, an original drug with antifungal activity against 33 clinically relevant mold isolates, including both azole-susceptible and -resistant Aspergillus. Using complete inhibition as an endpoint, interactions were indifferent for 32/33 isolates. An alternative 50% inhibition endpoint showed synergistic interactions for 14/33 isolates. Antagonism was absent.
Assuntos
Antifúngicos/farmacologia , Aspergilose/tratamento farmacológico , Aspergillus/efeitos dos fármacos , Fosforilcolina/análogos & derivados , Voriconazol/farmacologia , Aspergilose/microbiologia , Aspergillus/isolamento & purificação , Candida/efeitos dos fármacos , Farmacorresistência Fúngica , Sinergismo Farmacológico , Quimioterapia Combinada , Fusariose/tratamento farmacológico , Fusariose/microbiologia , Fusarium/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Fosforilcolina/farmacologia , Scedosporium/efeitos dos fármacosRESUMO
OBJECTIVES: Voriconazole, itraconazole and posaconazole are members of the azole family and widely used for the treatment of aspergillosis. They act by inhibiting the activity of the fungal Cyp51A enzyme. The emergence of environmental azole-resistant Aspergillus fumigatus strains raises major concerns for human health. METHODS: Recently, a new cyp51A-mediated resistance mechanism (namely TR46/Y121F/T289A) was described in clinical samples and patient-frequented environmental sites. In an azole-naive patient, we isolated an A. fumigatus strain that was not susceptible to voriconazole but was susceptible to itraconazole and posaconazole. RESULTS: A molecular analysis indicated a single Y121F substitution without the TR46 or T289A alterations, which to our knowledge has never been reported. Structure modelling and molecular dynamics offered an explanation for the resistance profile consistent with the structural differences between the three azoles. CONCLUSIONS: Taken together, these observations suggest an original mechanism conferring resistance to azoles mediated by cyp51A of environmental origin. This uncommon susceptibility pattern might represent a 'missing link' between the wild-type A. fumigatus and the fully azole-resistant strain harbouring the TR46/Y121F/T289A mutations.
Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/genética , Proteínas Fúngicas/genética , Itraconazol/farmacologia , Mutação de Sentido Incorreto , Triazóis/farmacologia , Voriconazol/farmacologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/isolamento & purificação , DNA Fúngico/química , DNA Fúngico/genética , Farmacorresistência Fúngica , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Since their introduction in the 2000s, echinocandin drugs have become widely used for the treatment and prophylaxis of invasive fungal infections and, notably, invasive candidiasis. Although cases of breakthrough candidiasis in patients receiving echinocandins have been reported, clinical failure during echinocandin treatment due to the acquisition of resistance by a normally susceptible Candida spp. isolate is considered rare. To date, no publications have been published correlating the use of echinocandins and the emergence of echinocandin resistance among Candida species. So, our goal is to report an initial analysis of echinocandin use in relation to the emergence of resistant Candida isolates. We report here a single-centre experience of the emergence of eight resistant isolates belonging to normally susceptible Candida species in six patients receiving echinocandins. We describe the context and analyse the use of echinocandins over the previous decade. For seven of these isolates, we identified FKS gene mutations involved in decreased susceptibility. Seven isolates were obtained in 2011, on the heels of a ten-fold increase in caspofungin use over the preceding decade. In contrast, in 2012, the use of echinocandins decreased in our institution by 19.5 % and, in that year, only one Candida-resistant isolate was detected, despite the stable global epidemiology of invasive candidaemia. This work underlines the necessity of improving the prescription of antifungal drugs. Improvement in the monitoring of strain susceptibility should also be considered in order to better detect the emergence of resistant or non-susceptible yeast strains.
Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Candidíase/microbiologia , Farmacorresistência Fúngica , Uso de Medicamentos , Equinocandinas/farmacologia , Idoso , Antifúngicos/uso terapêutico , Candida/isolamento & purificação , Candidíase/epidemiologia , Equinocandinas/uso terapêutico , Feminino , Hospitais , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , PrevalênciaRESUMO
Trichosporon spp. have recently emerged as significant human pathogens. Identification of these species is important, both for epidemiological purposes and for therapeutic management, but conventional identification based on biochemical traits is hindered by the lack of updates to the species databases provided by the different commercial systems. In this study, 93 strains, or isolates, belonging to 16 Trichosporon species were subjected to both molecular identification using IGS1 gene sequencing and matrix-assisted laser desorption ionisation-time-of-flight (MALDI-TOF) analysis. Our results confirmed the limits of biochemical systems for identifying Trichosporon species, because only 27 (36%) of the isolates were correctly identified using them. Different protein extraction procedures were evaluated, revealing that incubation for 30 min with 70% formic acid yields the spectra with the highest scores. Among the six different reference spectra databases that were tested, a specific one composed of 18 reference strains plus seven clinical isolates allowed the correct identification of 67 of the 68 clinical isolates (98.5%). Although until recently it has been less widely applied to the basidiomycetous fungi, MALDI-TOF appears to be a valuable tool for identifying clinical Trichosporon isolates at the species level.
Assuntos
Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Trichosporon/química , Trichosporon/classificação , Tricosporonose/diagnóstico , Tricosporonose/microbiologia , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Humanos , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Trichosporon/isolamento & purificaçãoRESUMO
BACKGROUND: Cutaneous leishmaniasis (CL) is a disfiguring but not life-threatening disease. Because antileishmanial drugs are potentially toxic, the World Health Organization (WHO) recommends simple wound care or local therapy as first-line treatment, followed or replaced by systemic therapy if local therapy fails or cannot be performed. METHODS: To determine the feasibility and impact of the recommended approach, we analyzed the results of a centralized referral treatment program in 135 patients with parasitologically proven CL. RESULTS: Infections involved 10 Leishmania species and were contracted in 29 different countries. Eighty-four of 135 patients (62%) were initially treated without systemic therapy. Of 109 patients with evaluable charts, 23 of 25 (92%) treated with simple wound care and 37 of 47 (79%) treated with local antileishmanial therapy were cured by days 42-60. In 37 patients with large or complex lesions, or preexisting morbidities, or who had not been cured with local therapy, the cure rate with systemic antileishmanial agents was 60%. Systemic adverse events were observed in 15 patients, all receiving systemic therapy. CONCLUSIONS: In this population of CL patients displaying variable degrees of complexity and severity, almost two-thirds of patients could be initially managed without systemic therapy. Of these, 60 were cured before day 60. The WHO-recommended stepwise approach favoring initial local therapy therefore resulted in at least 44% of all patients being cured without exposure to the risk of systemic adverse events. Efforts are needed to further simplify local therapy of CL and to improve the management of patients with complex lesions and/or preexisting comorbidities.
Assuntos
Antiprotozoários/uso terapêutico , Bandagens , Leishmaniose Cutânea/terapia , Viagem , Administração Tópica , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Adulto JovemRESUMO
Echinocandin drugs are widely used for the treatment of candidemia. Resistance is considered rare, and only a few cases of breakthrough candidiasis in patients receiving echinocandin have been reported worldwide. We report here for the first time a Candida kefyr isolate that acquired echinocandin resistance very rapidly after the initiation of caspofungin treatment for candidemia. We characterized the FKS gene mutation responsible for the resistance via the comparison of isolates sampled before and during treatment.
Assuntos
Antifúngicos/efeitos adversos , Candida/isolamento & purificação , Candidemia/microbiologia , Candidíase/microbiologia , Farmacorresistência Fúngica , Equinocandinas/efeitos adversos , Proteínas Fúngicas/genética , Glucosiltransferases/genética , Kluyveromyces/isolamento & purificação , Sequência de Aminoácidos , Candida/efeitos dos fármacos , Candida/genética , Candidemia/tratamento farmacológico , Candidíase/tratamento farmacológico , Caspofungina , Evolução Fatal , Feminino , Humanos , Kluyveromyces/efeitos dos fármacos , Kluyveromyces/genética , Lipopeptídeos , Pessoa de Meia-Idade , Dados de Sequência Molecular , MutaçãoRESUMO
Methylchavicol (CH(3)-CV), an important aromatic constituent of different plants like tarragon and basils, has been shown to be carcinogenic by a mechanism yet unclear, although it has been reported that carcinogenicity of CH(3)-CV in rodent might be linked to its metabolic conversion into a genotoxic electrophilic metabolite generated through a two steps bioactivation pathway catalyzed by cytochrome P450 enzymes and sulfotransferases. The induction of carcinogenesis by certain agents has been associated with the generation of oxidative stress. The aim of the present study was to determine whether pure methylchavicol applied on a human hepatoma cell line, HepG2, could promote oxidative stress and might alter the expression of procarcinogenic biomarkers such as the drug-metabolizing enzyme (CYP2E1), the inducible form of nitric oxide synthase (iNOS) and might induce the expression of Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and Mn-SOD that control the redox equilibrium of the cells. CH(3)-CV was shown to cause a significant induction of oxidative stress, as revealed by luminol-dependent chemiluminescence (LDCL) and to alter dramatically the expression of CYP2E1, iNOS and Mn-SOD, indicating that the toxic effect of CH(3)-CV could be mediated through a nitric oxide dependent mechanism. Under similar experimental conditions, the extracts from tarragon, chervil and basil did not induce such biological changes. These results provide evidence that the generation of an oxidative stress may be a significant event occurring during CH(3)-CV-induced toxicity. It also suggests that natural extracts containing different amounts of CH(3)-CV (tarragon, chervil and basil) did not elicit such toxicity and might contain compounds able to counteract this detrimental property.
Assuntos
Anisóis/química , Anisóis/farmacologia , Oxidantes/química , Oxidantes/farmacologia , Plantas Medicinais/química , Derivados de Alilbenzenos , Artemisia/química , Biomarcadores , Sobrevivência Celular/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Radicais Livres/química , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Óxido Nítrico Sintase/metabolismo , Óxidos de Nitrogênio/metabolismo , Ocimum basilicum/química , Estresse Oxidativo/efeitos dos fármacosRESUMO
Malaria is still the world's major important parasitic disease and is responsible for the death of more people than any other communicable disease except tuberculosis. A major change in recent years has been the recognition that severe malaria, predominantly caused by Plasmodium falciparum, is a complex multi-system disorder presenting with a range of clinical features. Some surviving patients have an increased risk of neurological and cognitive deficits, behavioural difficulties and epilepsy, making cerebral malaria a leading cause of childhood neurodisability in the malaria transmission area. It is unclear how an intravascular parasite causes such brain injury. Understanding of these mechanisms is important to develop appropriate neuroprotective interventions. However, due to the high specificity of P. falciparum to the human host and to the fact that clinical studies in human are not always feasible, our knowledge about this syndrome mainly comes from autopsy studies which can only give us a limited view of this deadly syndrome. Efforts developed by the scientific community have shown that development of severe malaria probably results from a combination of parasite-specific factors such as adhesion and sequestration to the vascular endothelium, the release of bioactive molecules, together with host inflammatory responses and metabolic acidosis. Recent studies have shown that endothelial cells could play a central role in the onset of the severe malaria. Indeed, adhesion of parasitized erythrocytes to these cells could drive their activation, which could participate in the trigger of an immune response and haemostatic derangements. Moreover, death of endothelial cells could be at the origin of the blood-lung/brain barrier breakdown. Despite the efforts to find new mechanisms, which explain the physiopathology of severe malaria, research progress is slowed down by the lack of experimental models, which reproduce this complex multi-system disorder. In absence of a vaccine so far, the rapid diagnosis of the disease, an efficient treatment, a correct management and nursing care are the only weapons to control mortality due to P. falciparum. It is important to note that in the future, the treatment of severe malaria may involve adjuvant treatments in addition to a potent antimalarial drug. In the present review, we summarize both what is known and practically useful for a physician, and the most promising and current topics of research.
Assuntos
Malária Cerebral/terapia , Adulto , Animais , Antimaláricos/uso terapêutico , Criança , Efeitos Psicossociais da Doença , Modelos Animais de Doenças , Feminino , Haplorrinos , Humanos , Controle de Infecções , Malária Cerebral/complicações , Malária Cerebral/diagnóstico , Malária Cerebral/epidemiologia , Malária Cerebral/parasitologia , Camundongos , Plasmodium falciparum/fisiologia , Gravidez , PrognósticoRESUMO
Invasive aspergillosis (IA) is a major threat for immunocompromised patients. Diagnostic difficulties often delay specific treatment initiation, which increases mortality. Finding new biomarkers to improve and speed accurate diagnosis is thus vital. To investigate the ability of proteomic methods for discovering new biomarkers of IA, we used a DIGE approach to perform a proteomic analysis on both bronchoalveolar lavages (BAL) and sera at different time-points of infection in a mouse model of invasive pulmonary aspergillosis. Progression of the infection was monitored using a bioluminescent strain of Aspergillus fumigatus. Sera proteins were enriched using the ProteoMiner kit (Biorad). This method allowed us to identify a fungal protein, the A. fumigatus major allergen Asp f 2, in sera of mice one day after the infection. However, this fungal protein was not detected three days after the infection. Importantly, in BAL, this work provides evidence of an in vivo complement evasion mechanism through the cleavage of C3b into three fragments during aspergillosis. Finally, our results underlining the inflammatory host response to IA in both lung and blood compartments at different times of infection may provide new insights into the pathophysiology of this disease.
Assuntos
Antígenos de Fungos/sangue , Líquido da Lavagem Broncoalveolar/química , Proteínas Fúngicas/sangue , Aspergilose Pulmonar Invasiva/diagnóstico , Alérgenos/análise , Animais , Aspergillus fumigatus/imunologia , Hospedeiro Imunocomprometido , Aspergilose Pulmonar Invasiva/sangue , Aspergilose Pulmonar Invasiva/imunologia , Medições Luminescentes , Camundongos , Análise de Componente Principal , Proteômica , Proteína Amiloide A Sérica/análise , Componente Amiloide P Sérico/análise , Eletroforese em Gel Diferencial BidimensionalRESUMO
BACKGROUND: For years, the analysis of innate responses to the major mold pathogen Aspergillus fumigatus has been restricted to specialized cells, such as professional phagocytes. More recently, the contribution of the airway epithelial barrier has been assessed and studies have shown that it was able to sense and react to the Aspergillus infection, for example, by producing cytokines. METHODS: To further explore the reaction of the respiratory epithelium to the fungus, we analyzed the proteome response of a human bronchial epithelial cell line to Aspergillus infection using difference gel electrophoresis. We studied the protein pattern of BEAS-2B cell culture supernatant after interaction of the cells with Aspergillus during a 15-hour coculture. RESULTS: We found formerly unknown aspects of bronchial cell behavior during Aspergillus infection: bronchial cells are able to develop both cellular defense mechanisms (ie, thioredoxin system activation) and immune reactions (ie, lysosomal degranulation and cathepsin activation) in response to the fungal aggression. CONCLUSIONS: Bronchial epithelial cells appear to be a more important effector of antifungal defense than expected. Degranulation of lysosomal enzymes that might be responsible for both fungal growth inhibition and host cell damage suggests that inductors/inhibitors of these pathways may be potential targets of therapeutic intervention.
Assuntos
Aspergillus fumigatus/patogenicidade , Células Epiteliais/metabolismo , Interações Hospedeiro-Patógeno , Proteínas/metabolismo , Proteoma/análise , Linhagem Celular , Técnicas de Cocultura , Meios de Cultura/química , Eletroforese/métodos , Humanos , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/microbiologiaRESUMO
Congenital transmission of Toxoplasma gondii occurs mainly when a mother acquires the infection for the first time during pregnancy. It was recently shown that although early treatment of the primary infection during pregnancy has little or no impact on the fetomaternal transmission rate, it does reduce the incidence of sequelae in infected infants. Seroconversion is defined by the appearance of IgG. Commercial reagents continue to vary considerably in detecting low concentrations of antibodies, as during early seroconversion. We compared two routinely used immunoassays (IA) (Platelia and Elecsys Toxo IgG) and an indirect immunofluorescence assay (IIF) with a qualitative test based on immunoblot analysis (Toxo II IgG) (IB) to assess their abilities to diagnose seroconversion at its earliest stages. This prospective study was carried out between January and November 2010. It included 39 pregnant women with monthly follow-up who seroconverted during pregnancy. On first sera that were IgM positive but IgG negative (or equivocal) as detected by IA, IB diagnosed seroconversion twice as often as IIF (26/39 [66.7%] versus 13/39 [33.3%]; P < 0.001; χ(2) test). Serum samples were retaken 2 to 5 weeks later for the other 13 cases (IgG negative by IB on first serum). Seroconversion was demonstrated as follows: IB for 5 cases where IA remained negative or equivocal, IB and IIF for 5 cases where IA remained negative or equivocal, IA for 2 cases, and no method for 1 case (a third sample was necessary). In summary, IB permitted toxoplasmosis seroconversion diagnosis before other means in 92.3% of cases (36/39) and thus earlier therapeutic intervention.
Assuntos
Anticorpos Antiprotozoários/sangue , Técnicas de Laboratório Clínico/métodos , Immunoblotting/métodos , Complicações Infecciosas na Gravidez/diagnóstico , Toxoplasma/imunologia , Toxoplasmose/diagnóstico , Adulto , Diagnóstico Precoce , Feminino , Técnica Indireta de Fluorescência para Anticorpo/métodos , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Gravidez , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
Immunodiagnostic assays are commonly used to screen for maternal toxoplasmic seroconversion during pregnancy. The introduction to the market of a new highly sensitive IgG assay, the Elecsys Toxo IgG test, has resulted in discrepancy issues with other immunoassays because of a lack of standardisation. Western blot appears to be a good alternative gold standard to the dye test, as the latter is not routinely available. For the present prospective study, we compared the analytical performances of two immunoassays, Elecsys Toxo IgG (Roche Diagnostics) and Platelia Toxo IgG (Bio-Rad, Marnes la Coquette, France), to Toxo II IgG Western blot (LDBio, Lyon, France) using 231 consecutive sera with low or equivocal IgG titres. Of these 231 sera, 213 presented discrepancies, which showed the importance of a confirmation test. Of the Elecsys Toxo IgG-positive results, 100% were confirmed by the Western blot with a positive threshold of 30 IU/ml for Elecsys; in the equivocal area (1-30 IU/ml), Western blot is negative in 54% of cases. Our results suggest that the lower diagnostic cut-off of Platelia Toxo IgG should be further reduced. Our study indirectly confirms that monitoring, especially for pregnant women, must be done in the same laboratory using the same technique. The ability to diagnose very early seroconversion using Western blot merits further study.
Assuntos
Anticorpos Antiprotozoários/sangue , Western Blotting/métodos , Imunoglobulina G/sangue , Parasitologia/métodos , Toxoplasma/imunologia , Toxoplasmose/diagnóstico , Adulto , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , França , Humanos , Masculino , Gravidez , Estudos ProspectivosRESUMO
We report the direct genotyping analysis of Toxoplasma gondii in ocular samples collected from 20 patients, as well as associated clinical and epidemiological data. This work was aimed at better understanding the impact of genotypes of Toxoplasma gondii strains on toxoplasmic retinochoroiditis. For this purpose, we studied the aqueous humor (AH) or vitreous humor (VH) of 20 patients presenting with ocular toxoplasmosis (OT) in 2 hospitals in France. Genetic characterization was obtained with microsatellite markers in a multiplex PCR assay. In contrast to the results of previous studies, we found no association between atypical Toxoplasma gondii genotypes and the occurrence of OT. Considering the local epidemiological data, our OT patients seemed to be infected more frequently by ordinary type II strains found in the environment. In conclusion, direct genotyping of Toxoplasma gondii strains from aqueous or vitreous humor showed a predominance of the type II genotype in ocular toxoplasmosis; this may be due to a high exposure rate of this genotype in humans.
Assuntos
Humor Aquoso/parasitologia , Toxoplasma/classificação , Toxoplasma/genética , Toxoplasmose Ocular/parasitologia , Corpo Vítreo/parasitologia , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA de Protozoário/genética , Feminino , França/epidemiologia , Genótipo , Humanos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Epidemiologia Molecular , Reação em Cadeia da Polimerase , Toxoplasma/isolamento & purificação , Adulto JovemRESUMO
Ocular toxoplasmosis is the principal cause of posterior uveitis and a leading cause of blindness. Animal models are required to improve our understanding of the pathogenesis of this disease. The method currently used for the detection of retinal cysts in animals involves the observation, under a microscope, of all the sections from infected eyes. However, this method is time-consuming and lacks sensitivity. We have developed a rapid, sensitive method for observing retinal cysts in mice infected with Toxoplasma gondii. This method involves combining the flat-mounting of retina - a compromise between macroscopic observation and global analysis of this tissue - and the use of an avirulent recombinant strain of T. gondii expressing the Escherichia coli beta-galactosidase gene, visually detectable at the submacroscopic level. Single cyst unilateral infection was found in six out of 17 mice killed within 28 days of infection, whereas a bilateral infection was found in only one mouse. There was no correlation between brain cysts number and ocular infection.