Characterization and Multicentric Validation of a Common Standard for Toxoplasma gondii Detection Using Nucleic Acid Amplification Assays (original) (raw)
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A New Alternative In Vitro Method for Quantification of Toxoplasma gondii Infectivity
Journal of Parasitology, 2012
An in vitro method to determine the infectious potency of an unknown suspension of the protozoan parasite Toxoplasma gondii based on kinetics of host cells lysis was developed. Mic1-3KO a mutant strain of T. gondii RH tachyzoites was inoculated in 25cm 2 flasks containing a 90% confluent monolayer of human foreskin fibroblasts. Lysis kinetics was monitored for infection ratios ranging from 1:10 6 to 1:10; we defined 10 6 tachyzoites/ml 21 as the threshold value for parasite egress. Results allowed us to build a calibration curve relating the initial infection ratios to the time needed to reach 10 6 tachyzoites/ml 21. Finally, we validated the method using a known mixture of dead and live parasites. This method was found to estimate with accuracy the initial ratio of infection of the unknown parasite suspension. This easy-to-use method is reproducible and can be applied to any T. gondii tachyzoite RH strain, genetically modified or not. This method is also suitable for testing promising candidates for an effective live vaccine.
Journal of Clinical Microbiology, 2010
Although screening for maternal toxoplasmic seroconversion during pregnancy is based on immunodiagnostic assays, the diagnosis of clinically relevant toxoplasmosis greatly relies upon molecular methods. A problem is that this molecular diagnosis is subject to variation of performances, mainly due to a large diversity of PCR methods and primers and the lack of standardization. The present multicentric prospective study, involving eight laboratories proficient in the molecular prenatal diagnosis of toxoplasmosis, was a first step toward the harmonization of this diagnosis among university hospitals in France. Its aim was to compare the analytical performances of different PCR protocols used for Toxoplasma detection. Each center extracted the same concentrated Toxoplasma gondii suspension and tested serial dilutions of the DNA using its own assays. Differences in analytical sensitivities were observed between assays, particularly at low parasite concentrations (<2 T. gondii genomes per reaction tube), with "performance scores" differing by a 20-fold factor among laboratories. Our data stress the fact that differences do exist in the performances of molecular assays in spite of expertise in the matter; we propose that laboratories work toward a detection threshold defined for a best sensitivity of this diagnosis. Moreover, on the one hand, intralaboratory comparisons confirmed previous studies showing that rep529 is a more adequate DNA target for this diagnosis than the widely used B1 gene. But, on the other hand, interlaboratory comparisons showed differences that appear independent of the target, primers, or technology and that hence rely essentially on proficiency and care in the optimization of PCR conditions.
FEMS Microbiology Letters, 1998
In order to investigate the accuracy and practicability of the polymerase chain reaction (PCR) in the antenatal diagnosis of congenital toxoplasmosis, a collaborative study involving 15 European laboratories was performed under the auspices of the Biomed 2 Programme of the European Community. Each team received 12 aliquots (four negative, eight positive) of`artificial samples' made of amniotic fluid spiked with tachyzoites of the RH strain of Toxoplasma gondii. Each team performed its own PCR protocol (all were different). Nine of the 15 laboratories were able to detect a single parasite, but two of the 15 found all samples negative. Four of the 15 laboratories found one or more control samples to be falsely positive. This study highlights the lack of homogeneity between PCR protocols and performance and underlines the need for an external quality assurance 0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 -1 0 9 7 ( 9 8 ) 0 0 2 8 1 -X FEMS Microbiology Letters 165 (1998) 231^237 scheme which could provide`reference' samples that could be used by any laboratory wanting to establish and maintain an accurate diagnostic test based on PCR. z
Real-time PCR for quantitative detection of Toxoplasma gondii
Journal of clinical microbiology, 2000
The protozoan Toxoplasma gondii is one of the most common infectious pathogenic parasites and can cause severe medical complications in infants and immunocompromised individuals. We report here the development of a real-time PCR-based assay for the detection of T. gondii. Oligonucleotide primers and a fluorescence-labeled TaqMan probe were designed to amplify the T. gondii B1 gene. After 40 PCR cycles, the cycle threshold values (C(T)) indicative of the quantity of the target gene were determined. Typically, a C(T) of 25.09 was obtained with DNA from 500 tachyzoites of the T. gondii RH strain. The intra-assay coefficients of variation (CV) were 0.4, 0.16, 0.24, and 0.79% for the four sets of quadruplicate assays, with a mean interassay CV of 0.4%. These values indicate the reproducibility of this assay. Upon optimization of assay conditions, we were able to obtain a standard curve with a linear range (correlation coefficient = 0.9988) across at least 6 logs of DNA concentration. Hen...
European Journal of Clinical Microbiology & Infectious Diseases, 1996
To investigate the accuracy of the polymerase chain reaction (PCR) method for the detection of Toxoplasma gondii in clinical specimens, aliquots of amniotic fluid to which known amounts of Toxoplasma gondii DNA had been added were tested by five European Centres. Four laboratories were able to detect DNA at levels equivalent to ten tachyzoites or less, including two that detected DNA equivalent to a single parasite. Two laboratories erroneously found one of eight negative control samples to be positive. These findings confirm that the high level of sensitivity associated with the PCR method can be readily achieved under routine laboratory conditions, but they also underscore the potential for both false-positive and false-negative findings to occur. Furthermore, the results confirm the urgent need for an external quality assurance scheme to support laboratories employing PCR in a clinical context for the detection of Toxoplasma gondii.
Journal of Clinical Pathology, 2010
Background. The Research Institute for Tropical Medicine (RITM)-National Reference Laboratory (NRL) for Malaria and Other Parasites, mandated by the Department of Health-Philippines (DOH), administers an annual Proficiency Test (PT) in diagnostic medical parasitology to clinical laboratories throughout the Philippines through the National External Quality Assessment Scheme (NEQAS). The PT in Parasitology aims to monitor and evaluate the capability of Philippine laboratories in the identification of blood and intestinal parasites, and the estimation of malaria parasite density in malaria-infected blood films. As of 2018, participation in the NEQAS is an annual requirement by the Department of Health-Health Facilities and Services Regulatory Bureau (DOH-HFSRB) for each clinical laboratory to obtain a license to operate. Objective. This report aims to summarize the results of the PT for Parasitology and assess the performance of participating laboratories in malaria and fecal parasite microscopy from 2009 to 2015. Methodology. RITM-NRL oriented clinical laboratories in the NEQAS in 2008. Laboratories submitted their accomplished enrolment forms to RITM-NRL and paid fees to enroll in the PT in 2009 to 2015. Participating laboratories identified the species of malaria in blood films and the parasite/s in formalin-preserved fecal specimens. Estimation of parasite density in malaria blood films was performed as well. Results. One thousand five hundred forty (1,540) laboratories participated from 2009 to 2015. Mean and median scores in all seven years were below the cutoff score of 80. Schistosoma japonicum was the most difficult to identify with only 7.7% of laboratories having correct identification result. Majority of participants from 2010 to 2014 gave malaria parasite density estimates outside the acceptable range. Conclusion. Most participating laboratories performed poorly in the proficiency tests over the last seven years. Training and refresher courses for laboratorians are recommended in order to address the poor performance in the laboratory diagnosis of parasitic infections, especially the endemic and uncommon ones, in the country
Journal of Clinical Microbiology, 2014
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.
DNA extraction and PCR assays for detection of Toxoplasma gondii
APMIS, 2004
For detection of Toxoplasma gondii we compared the sensitivity of two different DNA extraction methods and three different PCR assays. Sensitivities of DNA extraction by QIAamp DNA mini Kit or MagNa pure followed by PCR, nested PCR and oligochromatography or Light Cycler PCR using either SYBR green chemistry or TaqMan probe were compared. No significant difference between extraction methods was found using pure T. gondii tachyzoites. Spiked blood samples, 10 4 to 10 parasites per sample, generated no difference in sensitivity between the two DNA extraction methods when analysed by nested PCR detected by oligochromatography or analysed by Light Cycler PCR TaqMan. In spiked blood samples Light Cycler PCR SYBR green was unable to detect the parasite and a reduction in sensitivity was observed with the TaqMan assay. Conventional PCR was more sensitive when DNA was extracted from the spiked samples using the QIAamp DNA mini Kit. Conventional and nested PCR were found to be more sensitive than Light Cycler PCR TaqMan using the QIAamp DNA mini Kit. It was not possible to use Light Cycler PCR SYBR green in blood samples. Conventional PCR was more sensitive for detection of T. gondii in spiked blood samples using QIAamp DNA mini Kit DNA extraction, suggesting that the choice of DNA extraction method may affect PCR assays differently.