RESUMO
The molecular detection of Toxoplasma gondii DNA is a key tool for the diagnosis of disseminated and congenital toxoplasmosis. This multicentric study from the Molecular Biology Pole of the French National Reference Center for toxoplasmosis aimed to evaluate Toxoplasma gondii Real-TM PCR kit (Sacace). The study compared the analytical and clinical performances of this PCR assay with the reference PCRs used in proficient laboratories. PCR efficiencies varied from 90% to 112%; linearity zone extended over four log units (R2 > 0.99) and limit of detection varied from 0.01 to ≤1 Tg/mL depending on the center. Determined on 173 cryopreserved DNAs from a large range of clinical specimens, clinical sensitivity was 100% [106/106; 95 confidence interval (CI): 96.5%-100%] and specificity was 100% (67/67; 95 CI: 94.6%-100%). The study revealed two potential limitations of the Sacace PCR assay: the first was the inconsistency of the internal control (IC) when added to the PCR mixture. This point was not found under routine conditions when the IC was added during the extraction step. The second is a lack of practicality, as the mixture is distributed over several vials, requiring numerous pipetting operations. Overall, this study provides useful information for the molecular diagnosis of toxoplasmosis; the analytical and clinical performances of the Sacace PCR kit were satisfactory, the kit having sensitivity and specificity similar to those of expert center methods and being able to detect low parasite loads, at levels where multiplicative analysis gives inconsistently positive results. Finally, the study recommends multiplicative analysis in particular for amniotic fluids, aqueous humor, and other single specimens.
Assuntos
Toxoplasma , Toxoplasmose Congênita , Toxoplasmose , Humanos , Toxoplasma/genética , Toxoplasmose/diagnóstico , Toxoplasmose/parasitologia , Toxoplasmose Congênita/diagnóstico , Toxoplasmose Congênita/parasitologia , DNA , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , DNA de Protozoário/genética , DNA de Protozoário/análiseRESUMO
BACKGROUND: Biomarker discovery remains a major challenge for predictive medicine, in particular, in the context of chronic diseases. This is true for the widespread protozoan Toxoplasma gondii which establishes long-lasting parasitism in metazoans, humans included. This microbe successively unfolds distinct genetic programs that direct the transition from high to low replicative potential inside host cells. As a slow-replicating cell, the T. gondii bradyzoite developmental stage persists enclosed in a cyst compartment within tissues including the nervous system, being held by a sustained immune equilibrium which accounts for the prolonged clinically silent phase of parasitism. Serological surveys indicate that nearly one third of the human population has been exposed to T. gondii and possibly host bradyzoites. Because any disruption of the immune balance drives the reverse transition from bradyzoite to fast replicating tachyzoite and uncontrolled growth of the latter, these people are at risk for life-threatening disease. While serological tests for discriminating recent from past infection are available, there is yet no immunogenic biomarker used in the serological test to allow ascertaining the presence of persistent bradyzoites. RESULTS: Capitalizing on genetically engineered parasites induced to produce mature bradyzoites in vitro, we have identified the BCLA/MAG2 protein being restricted to the bradyzoite and the cyst envelope. Using laboratory mice as relevant T. gondii host models, we demonstrated that BCLA/MAG2 drives the generation of antibodies that recognize bradyzoite and the enveloping cyst structure. We have designed an ELISA assay based on a bacterially produced BCLA recombinant polypeptide, which was validated using a large collection of sera from mice of different genetic backgrounds and infected with bcla+ or bcla-null cystogenic and non-cystogenic T. gondii strains. To refine the design of the ELISA assay, we applied high-resolution BCLA epitope mapping and identified a specific combination of peptides and accordingly set up a selective and sensitive ELISA assay which allowed the detection of anti-BCLA/MAG2 antibodies in the sera of human patients with various forms of toxoplasmosis. CONCLUSIONS: We brought proof of principle that anti-BCLA/MAG2 antibodies serve as specific and sensitive serological markers in the perspective of a combinatorial strategy for detection of persistent T. gondii parasitism.
Assuntos
Encéfalo/parasitologia , Toxoplasma/fisiologia , Toxoplasmose/diagnóstico , Animais , Biomarcadores/metabolismo , Doença Crônica , Camundongos , Testes Sorológicos , Toxoplasmose/parasitologia , Toxoplasmose/patologiaRESUMO
AIMS: We evaluate whether the serum and aqueous humour (AH) level of IgG anti-Hsp70.1 antibodies improved the biological diagnosis of ocular toxoplasmosis. METHODS AND RESULTS: In this prospective cross-sectional and multicentre study, serum and AH were collected at the time of active uveitis. Anti-Hsp70.1-antibody levels were determined by ELISA. Patients with confirmed (Group A1, n = 21) or suspected ocular toxoplasmosis (group A2, n = 30) were enrolled, as well as a control group of patients with cataract (group B, n = 42). Serum IgG anti-Hsp70.1 antibody levels were not significantly different within the group of uveitis patients (A1, n = 21 vs A2, n = 30, P = .8) and were significantly associated with the affected retinal zone (P = .006) and with the size of the retinal lesion (P = .03). Serum anti-Hsp70.1 antibody level was positive in 10 out of the 18 patients of group A2. Significant anti-Hsp-70.1 antibody level in AH was reported in only three patients (3 eyes) with confirmed ocular toxoplasmosis. CONCLUSION: While the level of IgG anti-Hsp-70.1 antibody in AH did not improve the laboratory diagnosis of ocular toxoplasmosis, its level in serum was of major significance for retinal damage diagnosis.
Assuntos
Anticorpos Antiprotozoários/análise , Humor Aquoso/imunologia , Proteínas de Choque Térmico HSP70/imunologia , Imunoglobulina G/análise , Toxoplasmose Ocular/imunologia , Adulto , Anticorpos Antiprotozoários/imunologia , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Toxoplasma/imunologia , Toxoplasmose Ocular/diagnóstico , Uveíte/diagnóstico , Uveíte/imunologiaRESUMO
Background Testing for anti-Toxoplasma immunoglobulin (Ig)M is of main importance in the context of pregnancy to promptly alert to an acute maternal infection prior to the detection of IgG and to identify infected newborns. Their absence helps exclude a recent maternal infection in the presence of IgG. Methods The performance of a Toxo IgM immunocapture prototype assay (bioMérieux, France) was compared with that of the VIDAS® Toxo IgM and the ARCHITECT® Toxo IgM (Abbott, Germany) assays at Grenoble and Lyon (France). A total of 1446 sera were sampled from (i) 1054 pregnant women found by routine workup to have no infection (n = 843), an acute infection (<4 months) (n = 28) or a chronic infection (>4 months) with residual (n = 120) or no IgM (n = 62); (ii) 50 three-serum panels sampled immediately after a maternal seroconversion; (iii) 242 samples taken in 41 children with a congenital toxoplasmosis (n = 122) and in 40 uninfected children (n = 120). Results In pregnant women, the overall agreement with the VIDAS® assay was 99.23% (CI: 99.16-99.27) and that with the ARCHITECT® assay was 99.14% (CI: 99.07-99.17). Sensitivity of the Toxo IgM prototype assay was 100% (CI: 87.66-100.00) and specificity was 99.64% (98.96-99.93). In acute maternal infections, IgM assays were detected as early with the prototype as with the other two. In the congenitally infected children, IgM were detected on their first sample in 25/40 with the prototype vs. 23/40 with the VIDAS® test. No uninfected child had positive IgM. Conclusion The prototype performed comparably to the ARCHITECT® and VIDAS® Toxo IgM assays for the diagnosis of maternal and congenital toxoplasmosis.
Assuntos
Anticorpos Antiprotozoários/sangue , Imunoglobulina M/sangue , Complicações Parasitárias na Gravidez/diagnóstico , Toxoplasmose Congênita/diagnóstico , Anticorpos Antiprotozoários/imunologia , Feminino , Humanos , Imunoglobulina M/imunologia , Testes Imunológicos/métodos , Gravidez , Complicações Parasitárias na Gravidez/sangue , Toxoplasma/imunologia , Toxoplasmose Congênita/sangueRESUMO
The delayed-release tablet formulation of posaconazole (POS-tab) results in higher plasma POS trough concentrations (Cmin) than the oral suspension (POS-susp), which raises the question of the utility of therapeutic drug monitoring (TDM). We aimed to compare the variability of the POS Cmin for the two formulations and identify determinants of the POS-tab Cmin and its variability. Demographic, biological, and clinical data from 77 allogeneic hematopoietic stem cell transplant patients (874 Cmin) treated with POS-tab (n = 41), POS-susp (n = 29), or both (n = 7) from January 2015 to December 2016 were collected retrospectively. Interpatient and within-subject coefficients of variation (CVs) of the Cmin adjusted to dose (D) were calculated for each formulation. Between-group comparisons were performed using a linear mixed effects model. The POS Cmin was higher for the tablet than for the suspension (median [25th-75th percentile]: 1.8 [1.2-2.4] mg/liter versus 1.2 [0.7-1.6] mg/liter, P < 0.0001). Interpatient CVs for the tablet and suspension were 60.8 versus 63.5% (P = 0.7), whereas within-subject CVs were 39.7 and 44.9%, respectively (P = 0.3). Univariate analysis showed that age and treatment by POS-tab were significantly and positively associated with the POS Cmin, whereas diarrhea was associated with a diminished POS Cmin Multivariate analysis identified treatment with POS-tab and diarrhea as independent factors of the POS Cmin, with a trend toward a lower impact of diarrhea during treatment with POS-tab (P = 0.07). Despite increased POS exposure with the tablet formulation, the variability of the POS Cmin was not significantly lower than that of the suspension. This suggests that TDM may still be useful to optimize tablet POS therapy.
Assuntos
Antifúngicos/farmacocinética , Monitoramento de Medicamentos/métodos , Transplante de Células-Tronco Hematopoéticas , Micoses/prevenção & controle , Triazóis/farmacocinética , Administração Oral , Adulto , Fatores Etários , Idoso , Análise de Variância , Antifúngicos/sangue , Antifúngicos/farmacologia , Diarreia/fisiopatologia , Esquema de Medicação , Feminino , Neoplasias Hematológicas/sangue , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Micoses/sangue , Micoses/microbiologia , Estudos Retrospectivos , Fatores de Risco , Suspensões , Comprimidos , Transplante Homólogo , Triazóis/sangue , Triazóis/farmacologiaRESUMO
Success of anti-infective therapy is a major challenge in some patients given anatomo-physiological changes and genetic variations. In this case anecdote, we report the management strategy of a patient suffering from chronic pulmonary aspergillosis in a context of anorexia nervosa and genetic polymorphism.
Assuntos
Anorexia Nervosa/fisiopatologia , Antifúngicos/farmacocinética , Azóis/farmacocinética , Citocromo P-450 CYP2C19/genética , Aspergilose Pulmonar/tratamento farmacológico , Administração Oral , Adulto , Anorexia Nervosa/complicações , Antifúngicos/administração & dosagem , Azóis/administração & dosagem , Citocromo P-450 CYP2C19/metabolismo , Feminino , Humanos , Absorção Intestinal/fisiologia , Testes Farmacogenômicos , Polimorfismo de Nucleotídeo Único , Aspergilose Pulmonar/complicações , Resultado do TratamentoRESUMO
Transplantation activity is increasing, leading to a growing number of patients at risk for toxoplasmosis. We reviewed toxoplasmosis prevention practices, prevalence, and outcomes for hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT; heart, kidney, or liver) patients in Europe. We collected electronic data on the transplant population and prevention guidelines/regulations and clinical data on toxoplasmosis cases diagnosed during 2010-2014. Serologic pretransplant screening of allo-hematopoietic stem cell donors was performed in 80% of countries, screening of organ donors in 100%. SOT recipients were systematically screened in 6 countries. Targeted anti-Toxoplasma chemoprophylaxis was heterogeneous. A total of 87 toxoplasmosis cases were recorded (58 allo-HSCTs, 29 SOTs). The 6-month survival rate was lower among Toxoplasma-seropositive recipients and among allo-hematopoietic stem cell and liver recipients. Chemoprophylaxis improved outcomes for SOT recipients. Toxoplasmosis remains associated with high mortality rates among transplant recipients. Guidelines are urgently needed to standardize prophylactic regimens and optimize patient management.
Assuntos
Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Órgãos/efeitos adversos , Toxoplasmose/epidemiologia , Toxoplasmose/etiologia , Adulto , Europa (Continente)/epidemiologia , Humanos , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Risco , TransplantadosRESUMO
Molecular diagnosis of congenital toxoplasmosis or disseminated toxoplasmosis is based mainly on PCR. The repeated DNA element rep529 has become the main DNA target used in most PCR methods, whether laboratory developed or commercial. In this multicenter study, we evaluated the Toxoplasma ELITe MGB (Elitech) commercial kit by comparison with three reference quantitative PCR assays (RAs) used routinely in three proficient laboratories of the French National Reference Center for Toxoplasmosis network, using Toxoplasma calibrated suspensions diluted to obtain a range of concentrations from 0.1 to 10,000 parasites/ml. These suspensions were extracted with either the DNA extraction kit (EXTRAblood; Elitech) recommended by the manufacturer or the QIAamp DNA minikit (Qiagen). The Toxoplasma ELITe MGB assay was also evaluated on a panel of 128 clinical samples, including 56 amniotic fluid samples, 55 placenta samples, and various other samples, of which 95 originated from patients with proven toxoplasmosis. The ELITe MGB assay amplified low-concentration replicates (<10 parasites/ml) of calibrated suspensions less frequently than the RAs of 2/3 laboratories. Additionally, the combination of EXTRAblood and Toxoplasma ELITe MGB yielded poorer sensitivity than the combination of QIAamp DNA minikit and ELITe MGB for low parasite concentrations (P < 0.001 for 1 parasite/ml). On clinical samples, the sensitivity and the specificity of the commercial assay were 89% and 100%, respectively. The sensitivity ranged from 79% (placenta samples) to 100% (amniotic fluid samples). Overall, this study shows that the Toxoplasma ELITe MGB assay is suitable for the diagnosis of toxoplasmosis from non-cell-rich or non-hemoglobin-rich samples and that the EXTRAblood kit is not optimal.
Assuntos
DNA de Protozoário/genética , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Toxoplasma/genética , Toxoplasmose Congênita/diagnóstico , Líquido Amniótico/parasitologia , Feminino , Humanos , Placenta/parasitologia , Gravidez , Sensibilidade e Especificidade , Toxoplasma/isolamento & purificação , Toxoplasmose Congênita/parasitologiaRESUMO
Toxoplasmosis is a life-threatening infection in immunocompromised patients (ICPs). The definitive diagnosis relies on parasite DNA detection, but little is known about the incidence and burden of disease in HIV-negative patients. A 3-year retrospective study was conducted in 15 reference laboratories from the network of the French National Reference Center for Toxoplasmosis, in order to record the frequency of Toxoplasma gondii DNA detection in ICPs and to review the molecular methods used for diagnosis and the prevention measures implemented in transplant patients. During the study period, of 31,640 PCRs performed on samples from ICPs, 610 were positive (323 patients). Blood (n = 337 samples), cerebrospinal fluid (n = 101 samples), and aqueous humor (n = 100 samples) were more frequently positive. Chemoprophylaxis schemes in transplant patients differed between centers. PCR follow-up of allogeneic hematopoietic stem cell transplant (allo-HSCT) patients was implemented in 8/15 centers. Data from 180 patients (13 centers) were further analyzed regarding clinical setting and outcome. Only 68/180 (38%) patients were HIV(+); the remaining 62% consisted of 72 HSCT, 14 solid organ transplant, and 26 miscellaneous immunodeficiency patients. Cerebral toxoplasmosis and disseminated toxoplasmosis were most frequently observed in HIV and transplant patients, respectively. Of 72 allo-HSCT patients with a positive PCR result, 23 were asymptomatic; all were diagnosed in centers performing systematic blood PCR follow-up, and they received specific treatment. Overall survival of allo-HSCT patients at 2 months was better in centers with PCR follow-up than in other centers (P < 0.01). This study provides updated data on the frequency of toxoplasmosis in HIV-negative ICPs and suggests that regular PCR follow-up of allo-HSCT patients could guide preemptive treatment and improve outcome.
Assuntos
Hospedeiro Imunocomprometido , Técnicas Microbiológicas , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase , Toxoplasma/isolamento & purificação , Toxoplasmose/epidemiologia , França/epidemiologia , Humanos , Prevalência , Estudos Retrospectivos , Análise de Sobrevida , Toxoplasma/genética , Toxoplasmose/diagnóstico , Toxoplasmose/parasitologia , Toxoplasmose/patologiaRESUMO
The detection of Toxoplasma gondii in amniotic fluid is an essential tool for the prenatal diagnosis of congenital toxoplasmosis and is currently essentially based on the use of PCR. Although some consensus is emerging, this molecular diagnosis suffers from a lack of standardization and an extreme diversity of laboratory-developed methods. Commercial kits for the detection of T. gondii by PCR were recently developed and offer certain advantages; however, they must be assessed in comparison with optimized reference PCR assays. The present multicentric study aimed to compare the performances of the Bio-Evolution T. gondii detection kit and laboratory-developed PCR assays set up in eight proficient centers in France. The study compared 157 amniotic fluid samples and found concordances of 99% and 100% using 76 T. gondii-infected samples and 81 uninfected samples, respectively. Moreover, taking into account the classification of the European Research Network on Congenital Toxoplasmosis, the overall diagnostic sensitivity of all assays was identical and calculated to be 86% (54/63); specificity was 100% for all assays. Finally, the relative quantification results were in good agreement between the kit and the laboratory-developed assays. The good performances of this commercial kit are probably in part linked to the use of a number of good practices: detection in multiplicate, amplification of the repetitive DNA target rep529, and the use of an internal control for the detection of PCR inhibitors. The only drawbacks noted at the time of the study were the absence of uracil-N-glycosylase and small defects in the reliability of the production of different reagents.
Assuntos
Reação em Cadeia da Polimerase , Kit de Reagentes para Diagnóstico , Toxoplasma/genética , Toxoplasmose Congênita/diagnóstico , Toxoplasmose Congênita/parasitologia , Líquido Amniótico/parasitologia , Estudos de Coortes , Feminino , Humanos , Ensaio de Proficiência Laboratorial , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Gravidez , Kit de Reagentes para Diagnóstico/normas , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
The molecular diagnosis of toxoplasmosis essentially relies upon laboratory-developed methods and suffers from lack of standardization, hence the large diversity of performances between laboratories. Moreover, quantifications of parasitic loads differ among centers, a fact which prevents the possible prediction of the severity of this disease as a function of parasitic loads. The objectives of this multicentric study performed in eight proficient laboratories of the Molecular Biology Pole of the French National Reference Center for Toxoplasmosis (NRC-T) were (i) to assess the suitability of a lyophilized preparation of Toxoplasma gondii as a common standard for use in this PCR-based molecular diagnosis and (ii) to make this standard available to the community. High-quality written procedures were used for the production and qualification of this standard. Three independent batches of this standard, containing concentrations ranging from 10(4) to 0.01 T. gondii genome equivalents per PCR, were first assessed: the linear dynamic range was ≥ 6 log, the intra-assay coefficients of variation (CV) from a sample containing 10 T. gondii organisms per PCR were 0.3% to 0.42%, and the interassay CV over a 2-week period was 0.76% to 1.47%. A further assessment in eight diagnostic centers showed that the standard is stable, robust, and reliable. These lyophilized standards can easily be produced at a larger scale when needed and can be made widely available at the national level. To our knowledge, this is the first quality control assessment of a common standard which is usable both for self-evaluation in laboratories and for accurate quantification of parasitic loads in T. gondii prenatal infections.
Assuntos
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/normas , Padrões de Referência , Toxoplasma/isolamento & purificação , Toxoplasmose/diagnóstico , França , Humanos , Carga Parasitária/métodos , Carga Parasitária/normas , Toxoplasma/genéticaRESUMO
A pan-fungal nucleic acid sequence based applification (NASBA) test was adapted and used for the first time to detect and quantify the level of filamentous fungi in environmental samples. Surface samples (n = 356) collected in a controlled air flow hematology ward were tested by mycological culture and the pan-fungal NASBA test. The overall percentage of agreement between culture and NASBA was 88%, the Kappa coefficient was equal to 0.61 (95%CI = [0.51; 0.72]). This pan-fungal NASBA test could be a promising tool to rapidly monitor the absence of molds in controlled environments.
Assuntos
Microbiologia Ambiental , Monitoramento Epidemiológico , Fungos/isolamento & purificação , Técnicas Microbiológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Replicação de Sequência Autossustentável/métodos , Animais , Fungos/classificação , Fungos/genética , Departamentos Hospitalares , HumanosRESUMO
We describe a small family outbreak of trichinellosis caused by the consumption of raw ham from a wild boar (Sus scrofa) hunted in the northern Alps of France in February 2022. Out of the six people, aged 3-69 years, who consumed the meat, three were confirmed cases, and three were suspected cases. Eosinophilia detected in four people was the hallmark that drove the diagnosis. Three patients presented with myalgia, two with intense and prolonged chest pain, and one with elevated troponin. One patient presented with dermographism during treatment. Anti-Trichinella IgG were detected in three symptomatic individuals after about ten weeks. One patient had negative serology and no symptoms, but was on long-term corticosteroid therapy. Trichinella britovi larvae (8.3 larvae/g) were detected in the wild boar meat remnants. Trichinellosis is rare in France, but this family outbreak is reminiscent of the circulation of this pathogen in wild animals, highlighting the need to inform hunters about the risk of infection linked to the consumption of raw meat of game animals, and about the need for veterinary inspection of game meat. The consumption of raw meat outside controlled circuits is a practice not devoid of risks, which justifies raising the awareness of hunters, doctors, and medical biologists.
Title: Un foyer de Trichinella britovi dans les Alpes du Nord françaises : investigation par un réseau local de prospection. Abstract: Nous décrivons une épidémie familiale de trichinellose causée par la consommation de jambon cru d'un sanglier (Sus scrofa) chassé dans le nord des Alpes françaises en février 2022. Sur les six personnes âgées de 3 à 69 ans qui ont consommé la viande, trois étaient des cas confirmés, et trois étaient des cas suspects. L'éosinophilie détectée chez quatre personnes a permis d'évoquer le diagnostic. Trois patients présentaient des myalgies, et deux des douleurs thoraciques intenses et prolongées dont un avec une troponine élevée. Un patient a présenté un dermographisme pendant le traitement. Des IgG anti-Trichinella ont été détectées chez trois individus symptomatiques après environ dix semaines. Un des patients avait une sérologie négative et aucun symptôme mais était sous corticothérapie au long cours. Des larves de Trichinella britovi (8,3 larves/g) ont été détectées dans les restes du jambon de sanglier incriminé. La trichinellose est rare en France, mais cette épidémie familiale rappelle la circulation de cet agent pathogène chez les animaux sauvages, qui nécessite d'informer les chasseurs sur les risques d'infections liés à la consommation de viande crue de gibier, et de préconiser un contrôle vétérinaire des viandes de gibier. La consommation de viande crue en dehors des circuits contrôlés est une pratique non dénuée de risques, qui justifie une sensibilisation des chasseurs, médecins et biologistes médicaux.
Assuntos
Trichinella , Triquinelose , Animais , Suínos , Triquinelose/epidemiologia , Triquinelose/veterinária , Carne , Surtos de Doenças , França/epidemiologia , Sus scrofaRESUMO
[This corrects the article DOI: 10.1371/journal.pntd.0009144.].
RESUMO
Toxoplasmosis is a worldwide infection that may cause severe disease and is regarded as a serious health problem in France. Detection of the parasite by molecular methods is crucial for diagnosing the disease. The extreme diversity of methods and performances of Toxoplasma PCR assays makes the use of commercial PCR kits an attractive alternative, as they offer a chance for standardization. We compared the performances of three molecular methods for the detection of Toxoplasma gondii DNA in amniotic fluid: a commercial method using nested PCR and two laboratory-developed methods, one using conventional PCR and the other one real-time PCR. This evaluation was based upon a T. gondii DNA serial dilution assay, three amniotic fluid samples spiked with T. gondii at different concentrations, and a clinical cohort of 33 amniotic fluid samples. The T. gondii DNA serial dilution assay showed a much lower sensitivity for the commercial kit than for the laboratory-developed methods. Moreover, out of 12 proven congenital toxoplasmosis cases, 91.7% were detected by the laboratory-developed assays, whereas only 50% were detected by the commercial kit. A lack of sensitivity of the method, partly due to the presence of PCR inhibitors, was the main drawback of the commercial method. This study emphasizes that commercial PCR diagnostic kits do not systematically perform better than carefully optimized laboratory-developed methods. There is a need for thorough evaluation of such kits by proficient groups, as well as for performance standards that commercial kits can be tested against to improve confidence in those selected by health care providers.
Assuntos
Técnicas de Diagnóstico Molecular/métodos , Toxoplasma/isolamento & purificação , Toxoplasmose Congênita/diagnóstico , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Feminino , França , Humanos , Lactente , Recém-Nascido , Reação em Cadeia da Polimerase , Gravidez , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Toxoplasma/genéticaRESUMO
BACKGROUND: IgG detection to determine immune status to Toxoplasma gondii infection and seroconversion mainly relies on ELISA techniques and, if necessary, on a confirmatory test, Western blot. This study evaluated the performance of the recomLine Toxoplasma IgG immunoblot (IB-recomLine) (Mikrogen) as a confirmatory test on a large number of sera. A total of 171 sera were selected (113 patients) and had previously been analyzed by two ELISA tests, ARCHITECT (Abbott) and VIDAS (bioMérieux) ± LDBIO-Toxo II IgG Western blot (WB-LDBIO) (LDBio). The sera were classified into three groups: group 1 included 50 sera without difficulty in interpreting the IgG results (patients with documented past infection or uninfected); group 2 included 47 sera with difficulty in interpreting the ELISA results; and group 3 included 74 sequential sera from 25 pregnant women with seroconversion. RESULTS: In group 1, overall IgG agreements were 94% and 90% with ARCHITECT and VIDAS, respectively. In group 2, low agreement was observed between IB-recomLine and WB-LDBIO, with eight false-positive and 13 false-negative results. In group 3, 4/13 seroconversions were detected earlier with IB-recomLine compared to other tests. CONCLUSIONS: IB-recomLine allowed for earlier diagnosis of toxoplasmic seroconversion compared to both ELISA tests and WB-LDBIO but led to insufficient performance to confirm the immune status when ELISA results were discordant or equivocal.
Title: Diagnostic sérologique de la toxoplasmose : évaluation du test commercial recomLine Toxoplasma IgG immunoblot (Mikrogen) basé sur des antigènes recombinants. Abstract: Contexte : La détection des IgG pour déterminer le statut immunitaire vis-à-vis de l'infection à Toxoplasma gondii et de la séroconversion repose principalement sur les techniques ELISA et, si nécessaire, sur un test de confirmation, le Western blot. Cette étude a évalué les performances de l'immunoblot recomLine Toxoplasma IgG (IB-recomLine) (Mikrogen) en tant que test de confirmation sur un grand nombre de sérums. Un total de 171 sérums ont été sélectionnés (113 patients) et ont été préalablement analysés par deux tests ELISA, ARCHITECT (Abbott) et VIDAS (bioMérieux) ± LDBIO-Toxo II IgG Western blot (WB-LDBIO) (LDBio). Les sérums ont été classés en trois groupes : le groupe 1 comprenait 50 sérums sans difficulté d'interprétation des résultats IgG (patients avec antécédents d'infection documentée ou non infectés); le groupe 2 comprenait 47 sérums avec des difficultés d'interprétation des résultats ELISA; le groupe 3 comprenait 74 sérums séquentiels de 25 femmes enceintes séroconverties. Résultats : Dans le groupe 1, les concordances globales des IgG étaient respectivement de 94 % et 90 % avec ARCHITECT et VIDAS. Dans le groupe 2, une faible concordance a été observée entre IB-recomLine et WB-LDBIO, avec huit faux positifs et 13 faux négatifs. Dans le groupe 3, 4/13 séroconversions ont été détectées plus tôt avec IB-recomLine par rapport aux autres tests. Conclusions : IB-recomLine a permis un diagnostic plus précoce de la séroconversion toxoplasmique par rapport aux tests ELISA et WB-LDBIO, mais a conduit à des performances insuffisantes pour confirmer le statut immunitaire lorsque les résultats ELISA étaient discordants ou équivoques.
Assuntos
Toxoplasma , Toxoplasmose , Humanos , Feminino , Gravidez , Anticorpos Antiprotozoários , Imunoglobulina G , Toxoplasmose/diagnóstico , Western Blotting , Imunoglobulina MRESUMO
The high sensitivity of the automated tests used for Toxoplasma gondii serology can yield false-positive IgM results due to aspecific reactions. On the other hand, specific therapy can delay IgG production and, therefore, the diagnosis of seroconversion. There is a need for confirmation tests to early detect seroconversions during pregnancy. We conducted a multicentre study to evaluate the diagnostic accuracy of the Toxo II IgG and a new, not yet commercialised Toxo II IgM western blot (WB) (LDBio diagnostics Lyon France) on 229 sera corresponding to 93 patients with seroconversions and 158 sera corresponding to 68 patients with nonspecific IgM. Sensitivity was 97.8% for IgM WB and 98.9% for IgG WB. Specificity was 89.7% and 100%, respectively. The concordance between IgM and IgG Toxo WB with the final diagnosis was very good, K = 0.89 and K = 0.99, respectively. In 5 cases (5.4%), the appearance of IgM, and in 55 cases (59.1%), the appearance of IgG was recorded by WB earlier than by traditional tests. In 10 cases (10.8%), IgM was detected after the traditional tests and in 2 cases (2.2%) for IgG. The association of IgG and IgM WB on the same sample not only detected all seroconversions but also correctly identified most of the false-positive results.
RESUMO
Real-time PCR plays a crucial role in the diagnosis of toxoplasmosis. In this multicenter study, the Toxoplasma RealCycler Universal assay was assessed for the diagnosis of toxoplasmosis by eight reference laboratories. DNAs from diverse clinical samples were included: 141 characterized samples from patients with different clinical forms of proven toxoplasmosis and 27 from patients without toxoplasmosis were tested in duplicate with the commercial assay. Final diagnosis was affirmed by each center by analysis of clinical settings and biological follow-up. Calibrated Toxoplasma gondii standards and 11 external quality control samples were also included. Discrepant results observed after the first run of commercial PCR were controlled by both reference and commercial PCR assays. Using the commercial assay, the detection threshold varied from 0.01 to 1 tachyzoites/mL, depending on the center. The relationship between crossing point and DNA concentration was linear over 4 log units (r2 > 0.99), and PCR efficiencies were satisfactory (89% to 104%). The results of the 11 external quality control samples were concordant after one retesting, but those for 3 clinical samples remained discrepant. Sensitivity and specificity were calculated at 97.8% (95% CI, 97.8%-100%) and 100% (95% CI, 87.2%-100%), respectively. Provided that PCRs are performed at least in duplicate to detect low parasitic loads, Toxoplasma RealCycler Universal PCR showed suitable performances to diagnose the different forms of toxoplasmosis.