ABSTRACT
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.
KEYWORDS: molecular diagnosis, Toxoplasma gondii, comparaison methods
INTRODUCTION
Toxoplasmosis is a parasitic infection due to a protozoan, Toxoplasma gondii, mainly acquired through the consumption of cysts in undercooked meat or oocysts in contaminated food or water (1). Infected people with T. gondii are most often asymptomatic, but in pregnant women, toxoplasmosis may be severe as tachyzoites are able to cross placenta, to infect the fetus and cause congenital toxoplasmosis (1). Infected individuals carry T. gondii cysts in their muscular and cerebral tissues. If immunosuppression occurs, reactivation of bradyzoites induces a life-threatening severe toxoplasmosis such as cerebral or disseminated toxoplasmosis (1). Detection of T. gondii forms in amniotic fluid (AF), cerebrospinal fluid (CSF), blood by microscopic examination, cell culture, and/or mice inoculation is poorly sensitive, and molecular diagnosis has revolutionized the management of congenital and opportunistic toxoplasmosis (2, 3). Today, a number of laboratory-developed/in-house PCRs and commercial real-time PCR assays have been described (4, 5). Toxoplasma gondii Real-TM (Sacace, Como, Italy) is a real-time PCR kit, targeting the Rep529 non-coding repetitive DNA sequence, for T. gondii DNA detection in clinical specimens including white blood cells of peripheral and umbilical cord blood, biopsy specimen, CSF, and AF. It should be stressed here that parasitic load is often very low in clinical specimens. Thus, in AF, median was found <10 T/mL (6). Knowledge of performance and especially sensitivity of the different existing method is of great importance. A wide range of laboratory-developed and commercialized methods are available for diagnosing toxoplasmosis (4). The aim of the French National Reference Center for Toxoplasmosis (http://cnrtoxoplasmose.chu-reims.fr/?lang=en, last accessed 08 January 2024) is to evaluate PCR tests for the molecular diagnosis of toxoplasmosis, in order to standardize performance at national level. To achieve this, it organizes external quality controls (EQCs), practice surveys, and method comparisons (4, 7–13). Here, a multicenter study involving eight laboratories (Centers 1–8) was performed to evaluate analytical and clinical performances of T. gondii real-TM (Sacace) for the diagnosis of congenital and opportunistic toxoplasmosis. We also report on the experience of a center (Center 9) that has been using this kit routinely since 2019.
MATERIALS AND METHODS
Study design
This multicentric study involved eight Parasitology-Mycology laboratories from French university hospitals with an expertise in the diagnosis of toxoplasmosis. All participating centers are accredited according to the ISO15189 norm. The eight centers performing prenatal diagnosis of congenital toxoplasmosis are accredited by the French Ministry of Health (Regional Health Agency). All centers participate in the external quality assessment (EQA) for T. gondii PCR organized by the molecular biology group of the French Toxoplasma gondii National Reference Center (4). In the first phase, four expert centers of this group (Centers 1–4) evaluated (i) the analytical sensitivity and PCR efficiency of the T. gondii Sacace PCR assay using a serial dilution from a calibrated T. gondii lyophilized standard and (ii) the technical agreement between their reference PCR and the T. gondii Sacace PCR using EQC assessments. In the second phase, Centers 1–8 included clinical specimens from hospital collections to evaluate clinical performance of the T. gondii Sacace PCR assay. In the third phase, we report the experience of one center (Center 9) that uses this kit in routine practice. As the other centers, this center has an expertise in the diagnosis of toxoplasmosis, has an agreement from the Ministry of Health (Regional Health Agency) to perform prenatal diagnosis for congenital toxoplasmosis, and is accredited according to the ISO15189 norm. This center responded with complete accuracy to all EQA sessions in which it has participated.
Spiked samples, EQC samples, and clinical specimens
Lyophilized standards consisting 2 mL of a 10,000 T. gondii genome/mL suspension (12) were extracted by Centers 1–4. Extracted DNA (see Table 1 for extraction methods) was then 10-fold diluted to obtain a range of concentrations from 10,000 to 0.01 T. gondii genome/mL. The highest concentration at 10,000 T. gondii genome/mL was tested in duplicate, the three following concentrations at 1,000, 100, and 10 T. gondii genome/mL were tested in triplicate, and the three lowest concentrations, until 0.01 T. gondii genome/mL, were analyzed in six wells each (see Table S1). Five EQC samples (TGDNA21-C1) provided by QCMD (Quality Control for Molecular Diagnostics, Scotland, United Kingdom) were assessed in this study and analyzed in duplicate by Centers 1–4. One was negative, one was a positive AF with a concentration estimated at 32 ± 13 T. gondii genome/mL, and the three other were replicates of a positive AF with a concentration estimated at 4 ± 3 T. gondii genome/mL. To check the absence of competitive effect induced by the internal control (IC) included in the T. gondii Sacace PCR assay, these samples were extracted as recommended by the manufacturer with and without the IC added before the extraction step. For the clinical specimens, since this retrospective study was based on cryopreserved previously extracted DNA and in the absence of manufacturer's recommendations, we decided to add 0.1 µL of IC per reaction in the T. gondii Sacace PCR mix, after preliminary tests (data not shown). In addition, eight centers retrospectively included a total of 173 clinical specimens that were preserved at −20°C as extracted DNA. In a previous work, we have demonstrated that this storage provides adequate preservation of T. gondii DNA for retrospective molecular analysis (14). Before inclusion in the study, patients and specimens were carefully categorized according to European Group for Blood and Marrow Transplantation Infectious Diseases Working Party classification (15) and to European Research Network on Congenital Toxoplasmosis group (16). All specimens were checked to have enough volume for the whole study.
TABLE 1.
Details of specimens, PCR methods, and signal analysis used in each center in routine diagnosis of toxoplasmosisb
| DNA extraction protocola | Number of specimens analyzed | Toxoplasma gondii PCR in routine diagnosis (Reference PCR) | Sacace PCR | ||||
|---|---|---|---|---|---|---|---|
| Center | Calibrated curves | EQC | Clinical specimens | Target PCR assay | PCR apparatus | PCR apparatus | |
| 1 | TNN | Yes | 5 | 14 | Rep529 In-house Fret PCR (17) | LC480 (Roche) | LC480 (Roche) |
| Proteinase K, boiled and protein precipitation (Promega) | |||||||
| 2 | QIAamp DNA Micro Kit (Qiagen) | Yes | 5 | 14 | Rep529 Bio-Evolution | CFX 96 (BioRad) | CFX 96 (BioRad) |
| Nuclisens easyMag (bioMérieux) | |||||||
| 3 | QIAamp DNA Mini Kit (Qiagen) | Yes | 5 | 48 | Rep529 In-house Fret PCR (17) | LC480 (Roche) | LC480 (Roche) |
| 4 | QIAamp DNA Mini Kit (Qiagen) | Yes | 5 | 22 | Rep529 Bio-Evolution | LC480 (Roche) | MX3005 (Stratagene) |
| QIAamp FPPE DNA tissue kit (Qiagen) | |||||||
| QIAsymphony (Qiagen) | |||||||
| 5 | QIAamp DNA Mini Kit (Qiagen) | No | 0 | 42 | Rep529 In-house PCR (18) | StepOne Plus (Applied Biosystems) | StepOne Plus (Applied Biosystems) |
| EZ1 (Qiagen) | |||||||
| 6 | MagNA Pure 96, MagNA Pure Compact (Roche) | No | 0 | 12 | Rep529 Bio-Evolution | Rotor Gene Q (Qiagen) | Rotor Gene Q (Qiagen) |
| Maxwell (Promega) | |||||||
| EZ1 (Qiagen) | |||||||
| 7 | QIAamp DNA Mini Kit (Qiagen) | No | 0 | 17 | Rep529 In-house PCR (17) | LC2.0 | LC480 (Roche) |
| 8 | EMAG (BioMerieux) | No | 0 | 14 | Rep529 Bio-Evolution | ABI7500 | ABI7500 (Applied Biosystems) |
| 9 | QIAamp DNA Mini Kit (Qiagen) | In routine practice | Rep529 T. gondii Sacace |
Rotor Gene Q (Qiagen) | Rotor Gene Q (Qiagen) | ||
Reference PCR methods
Laboratories processed specimens according to their own routine procedures for DNA extraction and PCR methods (Table 1; Table S1). Briefly, every center used a PCR method targeting Rep529, among which four centers used laboratory-developed PCR, and four used the Bio-Evolution PCR kit.
Toxoplasma gondii Real-TM PCR kit (Sacace)
The Toxoplasma gondii Real-TM PCR kit (Ref: P1-50FRT; Sacace Biotechnologies, Como, Italy) was performed according to the manufacturer's instructions. Of note, these instructions have some peculiarities: (i) the mastermix consists of mixing three reagents: PCR-Mix-1-FRT, PCR-mix-2-FRT, and the Polymerase TaqF. The kit also provides positive control [Pos Control DNA T. gondii/STI (С+)], negative control (C−), and internal control STI-87 (IC) vials. Each PCR reaction consists of 25 µL of master mix and 10 µL of eluted DNA; (ii) the amplification program includes a first step of five repeats without fluorescence detection, followed by 40 repeats with fluorescence detection. Clinical specimens were tested in duplicate using the T. gondii Sacace PCR assay containing 0.1 µL of IC per reaction. In case of discrepant results between duplicates, the specimen was regarded as positive, and the clinical file was investigated.
Data and statistical analysis
In the first phase of the study, analytical performances were evaluated by four centers and relied on the results obtained in the serial dilution assay, as well as EQC assessment. To calculate the PCR efficiencies, we used the quantitative cycle (Cq) values obtained in serial dilutions assays and plotted them on a logarithmic scale along with their corresponding concentrations. We generated a linear regression curve and calculated the slope of the trend line. PCR efficiency is calculated using the equation: Eff = [10(−1/slope)−1]*100. This also allowed us in determining the zone of linearity (regression analysis, R2). Limit of detection was approached and defined as the lowest concentration where >50% of the reactions remain positive (20). In the second phase, clinical performances were assessed by comparing qualitative results and quantitative Cq values obtained for clinical specimens between the commercial kit and the reference PCR of each laboratory. Qualitative results were analyzed through sensitivity and specificity calculations. Quantitative Cq values were compared performing Bland-Altman plots (21) (using Prism Software).
Ethical approval and informed consent
Clinical specimens were collected through the routine clinical activity of each participating center. As recommended in the ethical standards of the French Ethics Committee on Human experimentation and with the Helsinki Declaration of 1975, as revised in 2008, in agreement with the French Health Public Law (CSP Art L1121-1.1) for non-interventional retrospective study, no written or verbal informed consent to participate in the study from the patient was necessary. However, according to the law, written consent was obtained before any prenatal AF sampling. Patients were anonymized; the study does not include potentially identifying patient/participant information.
RESULTS
Analytical performances of T. gondii Sacace PCR assay
All PCR efficiencies measured in this study were ≥90. No difference was seen between the reference PCRs and the Sacace PCR assays performed in Centers 1–4 (Table 2; Fig. S1). With a R2 varying from 0.9954 to 1, linearity zone was similar whatever the center and the PCR methods used. The lowest concentration where >50% of the reactions remain positive (20) was similar and determined at ≤1 T. gondii genome/mL for all the centers except Center 1, which retrieved it at 0.01 T. gondii genome/mL (Table 2; Table S1; Fig. S1). All qualitative results of EQC samples were concordant (Table 2). Regarding quantitative results (Cq values), comparison of Cq values obtained using the T. gondii Sacace PCR assay and obtained using each reference PCR showed good agreement with ΔCqs ranging from 2 to 5.4, according to the center (Table 2; Table S1).
TABLE 2.
Analytical performance of T. gondii Sacace PCR kit: Determination of PCR efficiency and limit of detection and EQC exactitudec
| Slope | Efficiency | R2 | LoD (T/mL) | Eqc (N = 5) | ∆Cq | |
|---|---|---|---|---|---|---|
| Center 1 | ||||||
| Reference PCR | 3.1 | 108 | 0.9954 | 0.01 | Concordant | 3a |
| Sacace wo IC | 3.1 | 112 | 0.9981 | 0.01 | Concordant | 0.5b |
| Sacace w IC | 3.1 | 112 | 0.9986 | 0.01 | Concordant | |
| Center 2 | ||||||
| Reference PCR | 3.4 | 98 | 1.0000 | <1 | Concordant | 2a |
| Sacace wo IC | 3.6 | 90 | 0.9994 | <1 | Concordant | 0.3b |
| Sacace w IC | 3.5 | 94 | 0.9981 | <1 | Concordant | |
| Center 3 | ||||||
| Reference PCR | 3.5 | 94 | 0.9999 | <1 | Concordant | 2.6a |
| Sacace wo IC | 3.5 | 94 | 0.9997 | <1 | Concordant | 2b |
| Sacace w IC | 3.6 | 91 | 0.9986 | <1 | Concordant | |
| Center 4 | ||||||
| Reference PCR | 3.5 | 94 | 0.9999 | ≤1 | Concordant | 5.4a |
| Sacace wo IC | 3.3 | 100 | 0.9997 | <1 | Concordant | 0.6b |
| Sacace w IC | 3.5 | 92 | 0.9979 | <1 | Concordant | |
Mean difference between EQC Cq values obtained using reference method and using T. gondii Sacace PCR with IC introduced during extraction procedure, ∆Cq.
Mean difference between EQC Cq values obtained using T. gondii Sacace PCR with IC either introduced in the PCR mix or during extraction procedure, w: with, wo: without.
R2: regression analysis, LoD: limit of detection, EQC: external quality assessment.
Validation of IC introduced in the PCR mix instead of in the specimen before extraction
No impact in the addition of the IC in the PCR mix as compared to adding it before the extraction procedure was observed in serial dilution assays and EQC testing (Table 2; Table S1). This allowed us to continue the evaluation using this procedure in clinical specimens.
Clinical specimens
Among the 173 clinical specimens tested, 106 were sampled from patients diagnosed with congenital (n = 41), disseminated (n = 32), cerebral (n = 20), ocular (n = 12), or acute (n = 1) toxoplasmosis and 67 were sampled from patients without any T. gondii infection. More than a dozen of distinct matrixes were included in this study (Table 3; Table S1). Among the 173 clinical specimens tested, IC amplification failed in at least one of the duplicates from 15/173 (9%) specimens, corresponding to 24 out of 346 (6.9%) wells (Table S1, highlighted). Of these 15 specimens, seven were positive and eight were negative specimens. Puzzingly, a second run of the Sacace PCR assay allowed recovering a positive IC in both duplicate. Thus, 173/173 specimens presented interpretable results, and all presented qualitative results of T. gondii Sacace PCR assay in agreement with the reference PCR of each participating center. Thereby, all qualitative results obtained with the reference PCRs and the T. gondii Sacace PCR test in each center were concordant. Of note, among the 106 positive clinical specimens, eight specimens were found to be inconsistently positive in four centers that corresponded to specimens with a low parasite load close to the detection limit of the method, four with their reference PCRs, and six with the Sacace PCR kit, two of which were inconsistently positive with both methods (Center 2, clinical specimen 13; Center 6, clinical specimen 10) (Table 3; highlighted in Table S1). As compared to reference PCR, sensitivity and specificity of the T. gondii Sacace PCR were calculated at 100% (95 CI: 96.5%–100%) and 100% (95 CI: 94.6%–100%), respectively. Cq values obtained with both reference and commercial PCRs revealed an excellent concordance, excepted for 3/106 specimens in two centers that were outliers (Fig. 1). Bland-Altman analysis revealed bias ranging from 3.5 to 5.5 according to the centers (Fig. 1).
TABLE 3.
Characteristics of 173 clinical specimens included in this study
| Specimen nature | Number of specimens | ||||
|---|---|---|---|---|---|
| Toxoplasmosis | Positive Sacace resultsa | Absence of toxoplasmosis | Negative Sacace resultsa | Final results | |
| Congenital toxoplasmosis | Concordant | ||||
| Amniotic fluid (AF) | 17 (1) | 17 (2, 1) | 6 | 6 (1) | 100% |
| Placenta | 19 | 19 (1, 1) | 5 | 5 (1) | |
| Umbilical cord blood | 4 | 4 | 1 | 1 | |
| Fetal biopsy | 1 | 1 | 0 | 0 | |
| Cerebral toxoplasmosis | |||||
| Cerebrospinal fluid (CSF) | 16 (1) | 16 (2, 2) | 10 | 10 | 100% |
| Brain biopsy | 2 | 2 | 0 | 0 | |
| Blood | 2 | 2 | 0 | 0 | |
| Ocular toxoplasmosis | |||||
| Aqueous humor (AH) | 12 | 12 (2) | 3 | 3 (1) | 100% |
| Disseminated toxoplasmosis/Acute toxoplasmosis | |||||
| Blood/buffy coat | 26 (2) | 26 (2) | 25 | 25 (4) | 100% |
| Broncho alveolar lavage fluid (BALF)/sputum | 2 | 2 | 11 | 11 (1) | |
| Bone marrow | 2 | 2 | 0 | 0 | |
| Lymph node biopsy | 1 | 1 | 2 | 2 | |
| Biopsy/Abscess | 1 | 1 | 2 | 2 | |
| Other | 1 | 1 | 2 | 2 | |
| Total of clinical specimens | 106 | 106 | 67 | 67 | 100% |
In brackets, the numbers in bold corresponded to IC issue, and the numbers in italic corresponded to the inconstantly positive.
Fig 1.
Bland-Altman plots comparing the Cq values of the routine method and the Sacace PCR for each of the eight centers and for clinical specimens.
DISCUSSION
Molecular diagnosis of T. gondii is a highly valuable tool in the diagnosis of congenital and opportunistic toxoplasmosis. A sensitive test is of great importance to promptly start an effective treatment if necessary (22, 23). Commercial kits are now widely available and the French National Reference Center for T. gondii evaluates their performance as a network of independent experts. To our knowledge, no independent evaluation of the Sacace kit has been published; four centers confirmed the good analytical performances of the Sacace PCR assay on serial dilution assays and EQC samples. Eight centers confirmed the good clinical performances of the test, with sensitivity and specificity at 100% (95 CI: 96.5%–100%) and (95 CI: 94.6–100%), respectively, as compared to their reference PCRs. Of note, Cq values obtained with Sacace were “artificially” decreased as compared to those obtained with the reference PCRs, due to the fluorescence measurement, which begins after five amplification cycles in the T. gondii Sacace PCR protocol, then mean (±SD) delta Cq values were 3.2 (±1.5) and 4.4 (±0.8) concerning EQCs and clinical specimens, respectively. In this study, we worked on specimens from our routine activities, giving a wide range of parasite loads. Thus, four specimens were found to be at the sensitivity limit by the reference method of four centers. Using the T. gondii Sacace PCR, six Toxoplasma positive specimens were found inconstantly positive, two of which were also found inconsistently positive with the reference method (Table 3; highlighted in Table S1). This once again stresses the importance of duplicating PCRs to reduce the risk of false-negative results. The high percentage of negative IC observed in this study needs to be raised although the retrospective design of the present study did not allow to integrate the IC as recommended by the manufacturer, i.e., before extraction. However, in routine practice, this is not an uncommon practice, as DNA extractions are often shared between several bacteriology, virology, parasitology, and mycology parameters, and it should be noticed that not every IC can be added during the extraction step. One center uses this commercial PCR in routine practice for two years. They introduce the IC directly in the specimen before extraction procedure. In 2022, 697 PCRs were realized, of which 53 were T. gondii positive and only two specimens presented a negative IC, confirming that adding IC during extraction procedures is reliable. Another limitation of the use of the Sacace PCR assay in routine practice may be the test practicability. Here, the PCR mix consists, in addition, of three components (PCR-mix-1, PCR-mix-2-FRT, and the TaqF Polymerase) multiplying technical manipulations while other commercial kits offer a single ready-to-use master mix.
To conclude, the T. gondii Real-TM kit (Sacace) demonstrated high analytical and clinical performances for the diagnosis of toxoplasmosis, but with two potential limitations: inconsistency of IC when added in the PCR mixture and the fractionation of the different components of the master mix.
ACKNOWLEDGMENTS
We are grateful to Sylvie Douzou and France Joullié (Montpellier) for their technical help. We are grateful to the technical staff for their technical help (Sorbonne Université, AP-HP, Hôpital St Antoine, Laboratoire Commun de Génétique et Biologie Moléculaire). The authors would like to thank Valérie Martin, Mélanie Decara, and Florent Saragosa (Grenoble, France) for their technical support.
Extramural funding was received from "Santé publique France" through the French National Reference Center for leishmaniasis. The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Contributor Information
Juliette Guitard, Email: juliette.guitard@aphp.fr.
Yvon Sterkers, Email: yvon.sterkers@umontpellier.fr.
Bobbi S. Pritt, Mayo Clinic Minnesota, Rochester, Minnesota, USA
SUPPLEMENTAL MATERIAL
The following material is available online at https://doi.org/10.1128/jcm.01428-23.
Determination of PCR efficiency and limit of detection of T. gondii Sacace PCR kit. The Cq values obtained during serial dilution tests are plotted against concentration for the four participating centers, for reference PCR, Sacace PCR, and with (+) and without (wo) internal control (IC). Regression lines were obtained and the equations were used to determine PCR efficiency.
Analytical and clinical performances of Sacace T. gondii PCR kit. Determination of PCR efficiency and limit of detection of Sacace T. gondii PCR kit. Results of Sacace T. gondii PCR. IC: internal control, Eff: Efficiency of the PCR, R2: regression analysis, N: number, NA: not available. Highlighted in gray: Toxoplasma positive PCRs. Highlighted in orange: negative IC. Highlighted in yellow: inconstantly positive specimen.
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REFERENCES
- 1. Montoya JG, Liesenfeld O. 2004. Toxoplasmosis. Lancet 363:1965–1976. doi: 10.1016/S0140-6736(04)16412-X [DOI] [PubMed] [Google Scholar]
- 2. Robert MG, Brenier-Pinchart M-P, Garnaud C, Fricker-Hidalgo H, Pelloux H. 2021. Molecular diagnosis of toxoplasmosis: recent advances and a look to the future. Expert Rev Anti Infect Ther 19:1529–1542. doi: 10.1080/14787210.2021.1941867 [DOI] [PubMed] [Google Scholar]
- 3. Robert-Gangneux F, Dardé M-L. 2012. Epidemiology of and diagnostic strategies for toxoplasmosis. Clin Microbiol Rev 25:264–296. doi: 10.1128/CMR.05013-11 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Roux G, Varlet-Marie E, Bastien P, Sterkers Y, French National Reference Center for Toxoplasmosis Network . 2018. Evolution of Toxoplasma-PCR methods and practices: a French national survey and proposal for technical guidelines. Int J Parasitol 48:701–707. doi: 10.1016/j.ijpara.2018.03.011 [DOI] [PubMed] [Google Scholar]
- 5. Sterkers Y, Varlet-Marie E, Marty P, Bastien P, Toxoplasma-PCR A, Quality Control Group . 2010. Diversity and evolution of methods and practices for the molecular diagnosis of congenital toxoplasmosis in France: a 4-year survey. Clin Microbiol Infect 16:1594–1602. doi: 10.1111/j.1469-0691.2009.03101.x [DOI] [PubMed] [Google Scholar]
- 6. Romand S, Chosson M, Franck J, Wallon M, Kieffer F, Kaiser K, Dumon H, Peyron F, Thulliez P, Picot S. 2004. Usefulness of quantitative polymerase chain reaction in amniotic fluid as early prognostic marker of fetal infection with Toxoplasma gondii. Am J Obstet Gynecol 190:797–802. doi: 10.1016/j.ajog.2003.09.039 [DOI] [PubMed] [Google Scholar]
- 7. Brenier-Pinchart M-P, Filisetti D, Cassaing S, Varlet-Marie E, Robert-Gangneux F, Delhaes L, Guitard J, Yéra H, Bastien P, Pelloux H, Sterkers Y. 2022. Molecular diagnosis of toxoplasmosis: multicenter evaluation of the Toxoplasma RealCycler universal PCR assay on 168 characterized human samples. J Mol Diagn 24:687–696. doi: 10.1016/j.jmoldx.2022.03.009 [DOI] [PubMed] [Google Scholar]
- 8. Brenier-Pinchart M-P, Robert-Gangneux F, Accoceberry I, Pichard S, Garnaud C, Fricker-Hidalgo H, Lévêque MF, Hoarau G, Pelloux H, Bastien P, Sterkers Y, Varlet-Marie E. 2021. Multicenter comparative assessment of the TIB MolBiol Toxoplasma gondii detection kit and four laboratory-developed PCR assays for molecular diagnosis of toxoplasmosis. J Mol Diagn 23:1000–1006. doi: 10.1016/j.jmoldx.2021.05.010 [DOI] [PubMed] [Google Scholar]
- 9. Morelle C, Varlet-Marie E, Brenier-Pinchart M-P, Cassaing S, Pelloux H, Bastien P, Sterkers Y. 2012. Comparative assessment of a commercial kit and two laboratory-developed PCR assays for molecular diagnosis of congenital toxoplasmosis. J Clin Microbiol 50:3977–3982. doi: 10.1128/JCM.01959-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Filisetti D, Sterkers Y, Brenier-Pinchart M-P, Cassaing S, Dalle F, Delhaes L, Pelloux H, Touafek F, Varlet-Marie E, Yera H, Candolfi E, Bastien P. 2015. Multicentric comparative assessment of the bio-evolution Toxoplasma gondii detection kit with eight laboratory-developed PCR assays for molecular diagnosis of congenital toxoplasmosis. J Clin Microbiol 53:29–34. doi: 10.1128/JCM.01913-14 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Robert-Gangneux F, Brenier-Pinchart M-P, Yera H, Belaz S, Varlet-Marie E, Bastien P, Molecular Biology Study Group of the French National Reference Center for Toxoplasmosis . 2017. Evaluation of Toxoplasma ELITe MGB real-time PCR assay for diagnosis of toxoplasmosis. J Clin Microbiol 55:1369–1376. doi: 10.1128/JCM.02379-16 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Varlet-Marie E, Sterkers Y, Brenier-Pinchart M-P, Cassaing S, Dalle F, Delhaes L, Filisetti D, Pelloux H, Touafek F, Yera H, Bastien P. 2014. Characterization and multicentric validation of a common standard for Toxoplasma gondii detection using nucleic acid amplification assays. J Clin Microbiol 52:3952–3959. doi: 10.1128/JCM.01906-14 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Varlet-Marie E, Sterkers Y, Perrotte M, Bastien P, Molecular Biology working group of the French National Reference Centre for Toxoplasmosis . 2018. A new LAMP-based assay for the molecular diagnosis of Toxoplasmosis: Comparison with a proficient PCR assay. Int J Parasitol 48:457–462. doi: 10.1016/j.ijpara.2017.11.005 [DOI] [PubMed] [Google Scholar]
- 14. Delhaes L, Filisetti D, Brenier-Pinchart M-P, Pelloux H, Yéra H, Dalle F, Sterkers Y, Varlet-Marie E, Touafek F, Cassaing S, Bastien P. 2014. Freezing and storage at -20°C provides adequate preservation of Toxoplasma gondii DNA for retrospective molecular analysis. Diagn Microbiol Infect Dis 80:197–199. doi: 10.1016/j.diagmicrobio.2014.08.007 [DOI] [PubMed] [Google Scholar]
- 15. Martino R, Maertens J, Bretagne S, Rovira M, Deconinck E, Ullmann AJ, Held T, Cordonnier C. 2000. Toxoplasmosis after hematopoietic stem cell transplantation. Clin Infect Dis 31:1188–1195. doi: 10.1086/317471 [DOI] [PubMed] [Google Scholar]
- 16. Lebech M, Joynson DH, Seitz HM, Thulliez P, Gilbert RE, Dutton GN, Ovlisen B, Petersen E. 1996. Classification system and case definitions of Toxoplasma gondii infection in immunocompetent pregnant women and their congenitally infected offspring. Eur J Clin Microbiol Infect Dis 15:799–805. doi: 10.1007/BF01701522 [DOI] [PubMed] [Google Scholar]
- 17. Reischl U, Bretagne S, Krüger D, Ernault P, Costa J-M. 2003. Comparison of two DNA targets for the diagnosis of toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes. BMC Infect Dis 3:7. doi: 10.1186/1471-2334-3-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Robert-Gangneux F, Dupretz P, Yvenou C, Quinio D, Poulain P, Guiguen C, Gangneux J-P. 2010. Clinical relevance of placenta examination for the diagnosis of congenital toxoplasmosis. Pediatr Infect Dis J 29:33–38. doi: 10.1097/INF.0b013e3181b20ed1 [DOI] [PubMed] [Google Scholar]
- 19. Hohlfeld P, Daffos F, Costa JM, Thulliez P, Forestier F, Vidaud M. 1994. Prenatal diagnosis of congenital toxoplasmosis with a polymerase-chain-reaction test on amniotic fluid. N Engl J Med 331:695–699. doi: 10.1056/NEJM199409153311102 [DOI] [PubMed] [Google Scholar]
- 20. Sterkers Y, Varlet-Marie E, Cassaing S, Brenier-Pinchart M-P, Brun S, Dalle F, Delhaes L, Filisetti D, Pelloux H, Yera H, Bastien P. 2010. Multicentric comparative analytical performance study for molecular detection of low amounts of Toxoplasma gondii from simulated specimens. J Clin Microbiol 48:3216–3222. doi: 10.1128/JCM.02500-09 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. Martin Bland J, Altman D. 1986. Statistical methods for assessing agreement between two methods of clinical measurement. The Lancet 327:307–310. doi: 10.1016/S0140-6736(86)90837-8 [DOI] [PubMed] [Google Scholar]
- 22. Montoya JG, Remington JS. 2008. Management of Toxoplasma gondii infection during pregnancy. Clin Infect Dis 47:554–566. doi: 10.1086/590149 [DOI] [PubMed] [Google Scholar]
- 23. Dunay IR, Gajurel K, Dhakal R, Liesenfeld O, Montoya JG. 2018. Treatment of toxoplasmosis: historical perspective, animal models, and current clinical practice. Clin Microbiol Rev 31:e00057-17. doi: 10.1128/CMR.00057-17 [DOI] [PMC free article] [PubMed] [Google Scholar]
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Supplementary Materials
Determination of PCR efficiency and limit of detection of T. gondii Sacace PCR kit. The Cq values obtained during serial dilution tests are plotted against concentration for the four participating centers, for reference PCR, Sacace PCR, and with (+) and without (wo) internal control (IC). Regression lines were obtained and the equations were used to determine PCR efficiency.
Analytical and clinical performances of Sacace T. gondii PCR kit. Determination of PCR efficiency and limit of detection of Sacace T. gondii PCR kit. Results of Sacace T. gondii PCR. IC: internal control, Eff: Efficiency of the PCR, R2: regression analysis, N: number, NA: not available. Highlighted in gray: Toxoplasma positive PCRs. Highlighted in orange: negative IC. Highlighted in yellow: inconstantly positive specimen.