Assessment of Droplet Digital PCR for the Detection and Absolute Quantification of Toxoplasma gondii

Accurate tools for Toxoplasma gondii detection and quantification can be valuable for the early and effective management of toxoplasmosis. Droplet digital PCR (ddPCR) is a next-generation end-point PCR technique with high performance. The objective of the study was to evaluate the performance of ddPCR for the detection and absolute quantification of T. gondii. From January 2019 to October 2020, DNA samples collected at the Laboratory of Parasitology and Mycology of Pitié-Salpêtrière Hospital in Paris were retrospectively analyzed by ddPCR and real-time quantitative PCR (qPCR). To detect T. gondii with the best sensitivity possible, the REP-529 multicopy target was used. For absolute quantification of T. gondii, a specific single-copy target of α-tubulin was designed. T. gondii detection by ddPCR and qPCR was strongly correlated (R2 = 0.93), with a total concordance of 96.7% (n = 145/150). Quantification of T. gondii using ddPCR was successful for 15 of 35 samples showing a parasite load ≥170 copies/mL of DNA eluate using the α-tubulin target. The qPCR REP-529 quantification based on a standard curve was approximate and dependent on the strain genotype, which led to an estimate of parasite copy number 14- to 160-fold superior to the ddPCR result. In total, ddPCR is an effective molecular method for T. gondii detection that shows equivalent performance to qPCR. For robust T. gondii quantification, ddPCR is clearly more accurate than semiquantitative qPCR methods. Accurate tools for Toxoplasma gondii detection and quantification can be valuable for the early and effective management of toxoplasmosis. Droplet digital PCR (ddPCR) is a next-generation end-point PCR technique with high performance. The objective of the study was to evaluate the performance of ddPCR for the detection and absolute quantification of T. gondii. From January 2019 to October 2020, DNA samples collected at the Laboratory of Parasitology and Mycology of Pitié-Salpêtrière Hospital in Paris were retrospectively analyzed by ddPCR and real-time quantitative PCR (qPCR). To detect T. gondii with the best sensitivity possible, the REP-529 multicopy target was used. For absolute quantification of T. gondii, a specific single-copy target of α-tubulin was designed. T. gondii detection by ddPCR and qPCR was strongly correlated (R2 = 0.93), with a total concordance of 96.7% (n = 145/150). Quantification of T. gondii using ddPCR was successful for 15 of 35 samples showing a parasite load ≥170 copies/mL of DNA eluate using the α-tubulin target. The qPCR REP-529 quantification based on a standard curve was approximate and dependent on the strain genotype, which led to an estimate of parasite copy number 14- to 160-fold superior to the ddPCR result. In total, ddPCR is an effective molecular method for T. gondii detection that shows equivalent performance to qPCR. For robust T. gondii quantification, ddPCR is clearly more accurate than semiquantitative qPCR methods. Toxoplasmosis is an infectious disease caused by Toxoplasma gondii, a protozoan parasite.1Montoya J.G. Liesenfeld O. Toxoplasmosis.Lancet. 2004; 363: 1965-1976Abstract Full Text Full Text PDF PubMed Scopus (2658) Google Scholar Up to one-third of the world’s human population is infected by T. gondii. However, toxoplasmosis is considered a neglected disease in many countries.2Wallon M. Peyron F. Congenital toxoplasmosis: a plea for a neglected disease.Pathogens. 2018; 7: 25Crossref PubMed Scopus (50) Google Scholar The diagnosis of toxoplasmosis is challenging. Real-time quantitative PCR (qPCR) techniques are recommended for a precocious diagnosis of prenatal, neonatal, cerebral, disseminated, and retinochoroiditis infections.3Bastien P. Diagnosis molecular diagnosis of toxoplasmosis.Trans R Soc Trop Med Hyg. 2002; 96: S205-S215Abstract Full Text PDF PubMed Google Scholar Despite scarce and contradictory literature, parasitic load estimation by qPCR could prove useful as a prognostic marker of disease severity. Indeed, for congenital infections, the presence of clinical signs in fetuses has been correlated with a higher concentration of T. gondii parasites in amniotic fluid.4Romand S. Chosson M. Franck J. Wallon M. Kieffer F. Kaiser K. Dumon H. Peyron F. Thulliez P. Picot S. Usefulness of quantitative polymerase chain reaction in amniotic fluid as early prognostic marker of fetal infection with Toxoplasma gondii.Am J Obstet Gynecol. 2004; 190: 797-802Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar,5Costa J. Ernault P. Gautier E. Bretagne S. Prenatal diagnosis of congenital toxoplasmosis by duplex real-time PCR using fluorescence resonance energy transfer hybridization probes.Prenat Diagn. 2001; 21: 85-88Crossref PubMed Scopus (81) Google Scholar In immunocompromised patients, quantitative qPCR was useful for posttherapeutic follow-up to monitor the decrease in parasite loads.6Patrat-Delon S. Gangneux J.P. Lavoué S. Lelong B. Guiguen C. le Tulzo Y. Robert-Gangneux F. Correlation of parasite load determined by quantitative PCR to clinical outcome in a heart transplant patient with disseminated toxoplasmosis.J Clin Microbiol. 2010; 48: 2541-2545Crossref PubMed Scopus (39) Google Scholar,7Kupferschmidt O. Krüger D. Held T.K. Ellerbrok H. Siegert W. Janitschke K. Kupferschmidt O. Krüger D. Held T.K. Ellerbrok H. Siegert W. Janitschke K. Quantitative detection of Toxoplasma gondii DNA in human body fluids by TaqMan polymerase chain reaction.Clin Microbiol Infect. 2001; 7: 120-124Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar To date, routine T. gondii qPCR quantification methods are still approximate, with most of them using a standard curve from a biological sample spiked with a T. gondii reference strain.8Varlet-Marie E. Sterkers Y. Brenier-Pinchart M.P. Cassaing S. Dalle F. Delhaes L. Filisetti D. Pelloux H. Touafek F. Yera H. Bastien P. Characterization and multicentric validation of a common standard for Toxoplasma gondii detection using nucleic acid amplification assays.J Clin Microbiol. 2014; 52: 3952-3959Crossref PubMed Scopus (20) Google Scholar To increase sensitivity, REP-529, a repeated noncoding DNA sequence, is the standard molecular target used in France and most European countries.9Belaz S. Gangneux J.P. Dupretz P. Guiguen C. Robert-Gangneux F. A 10-year retrospective comparison of two target sequences, REP-529 and B1, for Toxoplasma gondii detection by quantitative PCR.J Clin Microbiol. 2015; 53: 1294-1300Crossref PubMed Scopus (50) Google Scholar, 10Reischl U. Bretagne S. Krüger D. Ernault P. Costa J.-M. Comparison of two DNA targets for the diagnosis of toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes.BMC Infect Dis. 2003; 3: 7Crossref PubMed Scopus (280) Google Scholar, 11Roux G. Varlet-Marie E. Bastien P. Sterkers Y. Evolution of Toxoplasma-PCR methods and practices: a French national survey and proposal for technical guidelines.Int J Parasitol. 2018; 48: 701-707Crossref PubMed Scopus (16) Google Scholar However, the number of copies of REP-529 varies 200- to 300-fold,12Homan W.L. Vercammen M. De Braekeleer J. Verschueren H. Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR.Int J Parasitol. 2000; 30: 69-75Crossref PubMed Scopus (574) Google Scholar and it may have an impact on quantification. The method of extraction and the efficacy of the qPCR assay may also cause result variability.11Roux G. Varlet-Marie E. Bastien P. Sterkers Y. Evolution of Toxoplasma-PCR methods and practices: a French national survey and proposal for technical guidelines.Int J Parasitol. 2018; 48: 701-707Crossref PubMed Scopus (16) Google Scholar,13Yera H. Ménégaut L. Brenier-Pinchart M.P. Touafek F. Bastien P. Dalle F. Cassaing S. Delhaes L. Filisetti D. Ménotti J. Pelloux H. Robert-Gangneux F. Sterkers Y. Varlet-Marie E. Evaluation of five automated and one manual method for Toxoplasma and human DNA extraction from artificially spiked amniotic fluid.Clin Microbiol Infect. 2018; 24: 1100.e7-1100.e11Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar Therefore, a rapid, accurate, and easy to standardize absolute quantification method is required. Droplet digital PCR (ddPCR) is a next-generation end-point PCR that exhibits high performance for the detection and absolute quantification of specific targets in various human biological samples,14Cao L. Cui X. Hu J. Li Z. Choi J.R. Yang Q. Lin M. Ying Hui L. Xu F. Advances in digital polymerase chain reaction (dPCR) and its emerging biomedical applications.Biosens Bioelectron. 2017; 90: 459-474Crossref PubMed Scopus (185) Google Scholar including major applications in oncology, particularly for liquid biopsies.15Denis J.A. Guillerm E. Coulet F. Larsen A.K. Lacorte J.-M. The Role of BEAMing and digital PCR for multiplexed analysis in molecular oncology in the era of next-generation sequencing.Mol Diagn Ther. 2017; 21: 587-600Crossref PubMed Scopus (33) Google Scholar,16Hudecova I. Digital PCR analysis of circulating nucleic acids.Clin Biochem. 2015; 48: 948-956Crossref PubMed Scopus (143) Google Scholar It is promising for absolute quantification of bacterial, viral, and parasite loads in clinical samples without a standard curve and for the calibration of reference standards for qPCR assays.17Kuypers J. Jerome K.R. Applications of digital PCR for clinical microbiology.J Clin Microbiol. 2017; 55: 1621-1628Crossref PubMed Scopus (135) Google Scholar, 18Li H. Bai R. Zhao Z. Tao L. Ma M. Ji Z. Jian M. Ding Z. Dai X. Bao F. Liu A. Application of droplet digital PCR to detect the pathogens of infectious diseases.Biosci Rep. 2018; 38BSR20181170Crossref Scopus (121) Google Scholar, 19Lei S. Chen S. Zhong Q. Digital PCR for accurate quantification of pathogens: principles, applications, challenges and future prospects.Int J Biol Macromol. 2021; 184: 750-759Crossref PubMed Scopus (20) Google Scholar In parasitology, although successfully applied for a range of protozoa and helminths,20Pomari E. Piubelli C. Perandin F. Bisoffi Z. Digital PCR: a new technology for diagnosis of parasitic infections.Clin Microbiol Infect. 2019; 25: 1510-1516Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar it has never been evaluated for T. gondii diagnosis in humans. This study aimed to evaluate the performance of ddPCR for the detection and absolute quantification of T. gondii. A first assay was aimed at detecting T. gondii with the best sensitivity possible using the REP-529 multicopy target, and a second assay was aimed at absolute ddPCR quantification of T. gondii, due to a specific single-copy region of α-tubulin. From January 2019 to October 2020, DNA samples (n = 150) were collected at the Laboratory of Parasitology and Mycology of Pitié-Salpêtrière Hospital in Paris and stored at −80°C following the routine diagnosis of toxoplasmosis. The DNA samples were obtained from blood (n = 98, including one placental cord blood), amniotic fluid (AF) (n = 10), placenta (n = 4), cerebrospinal fluid (CSF) (n = 7), aqueous humour (AH) (n = 20), and bronchoalveolar fluid (BAL) (n = 7). Four quality controls (2 AF and 2 blood samples) were included from the molecular biology associated laboratory of the National Reference Centre for Toxoplasmosis (Montpellier, France). Two reference strains of T. gondii were used: the ME49 strain (TgA 00001, type II) provided by the Biological Resource Centre for Toxoplasma (http://www.toxocrb.com, last accessed January 2023) and the RH strain (type I) provided by the Montpellier laboratory. The ME49 strain was obtained from an infected mouse brain, whereas the RH strain was obtained from lyophilized spiked AF. The RH strain was used for the elaboration of a standard curve (initial concentration of 105/mL of T. gondii genome equivalents), Production of the RH stock suspensions has been described and validated elsewhere.8Varlet-Marie E. Sterkers Y. Brenier-Pinchart M.P. Cassaing S. Dalle F. Delhaes L. Filisetti D. Pelloux H. Touafek F. Yera H. Bastien P. Characterization and multicentric validation of a common standard for Toxoplasma gondii detection using nucleic acid amplification assays.J Clin Microbiol. 2014; 52: 3952-3959Crossref PubMed Scopus (20) Google Scholar Briefly, tachyzoites were grown in vitro by serial passage in human foreskin fibroblast monolayers in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum using standard procedure. Harvested tachyzoites were washed twice, resuspended in sterile RPMI 1640 medium, and counted in a hemocytometer. Means and SDs were calculated from 10 values, and the parasites were then diluted in AF to obtain 105/mL of T. gondii genome equivalents. The sample volume of blood was 200 μL. For CSF, BAL, AF, placenta, and AH samples, a centrifugation step at 11,180 × g for 2 to 5 minutes was implemented before DNA extraction. For the AH samples, the resultant supernatant was discarded and 200 μL of phosphate-buffered saline was added to the centrifugation pellet. For the other samples, the pellet was resuspended in 200 μL of initial matrix. DNA was manually isolated using a Qiamp DNA mini kit (Qiagen, Hildesheim, Germany) or an Emag (bioMérieux, Paris, France) automatized DNA extraction following the manufacturer’s recommendations. The elution volume was 200 μL for manual DNA extraction or 100 μL for automated DNA extraction. DNA concentrations were determined using a Qubit double-stranded DNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA). The method of extraction used for each sample is detailed in Supplemental Table S1. T. gondii detection was performed by amplification of REP-529, a noncoding repeat element sequence located on chromosome IV (http://www.ncbi.nlm.nih.gov/genbank; accession number AF487550),10Reischl U. Bretagne S. Krüger D. Ernault P. Costa J.-M. Comparison of two DNA targets for the diagnosis of toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes.BMC Infect Dis. 2003; 3: 7Crossref PubMed Scopus (280) Google Scholar,12Homan W.L. Vercammen M. De Braekeleer J. Verschueren H. Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR.Int J Parasitol. 2000; 30: 69-75Crossref PubMed Scopus (574) Google Scholar using previously described primers and probe.21Bourdin C. Busse A. Kouamou E. Touafek F. Bodaghi B. le Hoang P. Mazier D. Paris L. Fekkar A. PCR-based detection of Toxoplasma gondii DNA in blood and ocular samples for diagnosis of ocular toxoplasmosis.J Clin Microbiol. 2014; 52: 3987-3991Crossref PubMed Scopus (48) Google Scholar Final concentrations were 0.5 μmol/L for primers and 0.2 μmol/L for probe. In each reaction, the sample volume of DNA extract was 5 μL if the DNA concentration was ≥2 ng/μL and 10 μL if <2 ng/μL. REP-529 was amplified using a 7500 Fast Real-Time PCR instrument (Applied Biosystems, Waltham, MA). The cycling conditions were as follows: 95°C for 20 seconds followed by 45 cycles of 2 seconds at 95°C and 20 seconds at 68°C. All the samples showing a characteristic amplification curve and an amplification threshold cycle (CT) <40 were deemed positive. Negative (no DNA template) and positive controls (ME49 and RH strains) were applied in each experiment. All experiments were repeated three times. ddPCR was performed to detect and quantify T. gondii using a QX200 Droplet Digital PCR system (Bio-Rad Laboratories, Hercules, CA). For ddPCR REP-529, the same volume of DNA extract as that used for qPCR REP-529 was used in each reaction for adequate comparisons. For ddPCR α-tubulin, a volume of 10 μL of DNA extract was used to gain sensitivity. Final concentrations of 2× ddPCR supermix (no dUTP) (Bio-Rad) were 900 nmol/L for primers and 250 nmol/L for probes in a total volume of ddPCR mix of 20 μL. The cycling conditions were as follows: 95°C for 10 minutes (polymerase activation), followed by 45 cycles of 95°C for 30 seconds, 60°C (REP-529), or 55°C (α-tubulin, see primers and probe below) for 60 seconds (denaturation and elongation), and 98°C for 10 minutes (droplet consolidation), with a final hold at 12°C (cooling and preservation). The data were analyzed using Quanta Soft software Pro version 1.7 (Bio-Rad) with the thresholds set based on the results obtained during the validation method (see Results section). For a normalization concern, quantification of T. gondii was expressed in copy numbers per milliliter of eluate, which was determined by multiplication of the mean copy numbers per microliters of reaction (absolute quantification provided by the ddPCR system) by a factor of ×1000. Quantification with the α-tubulin gene was performed for all samples, with an REP-529–positive signal obtained by real-time PCR and/or digital PCR. All experiments were repeated three times. The validation method was performed following the Digital MIQE Guidelines.22Whale A.S. de Spiegelaere W. Trypsteen W. Nour A.A. Bae Y.-K. Benes V. Burke D. Cleveland M. Corbisier P. Devonshire A.S. Dong L. Drandi D. Foy C.A. Garson J.A. He H.-J. Hellemans J. Kubista M. Lievens A. Makrigiorgos M.G. Milavec M. Mueller R.D. Nolan T. O’Sullivan D.M. Pfaffl M.W. Rödiger S. Romsos E.L. Shipley G.L. Taly V. Untergasser A. Wittwer C.T. Bustin S.A. Vandesompele J. Huggett J.F. The digital MIQE guidelines update: minimum information for publication of quantitative digital PCR experiments for 2020.Clin Chem. 2020; 66: 1012-1029Crossref PubMed Scopus (165) Google Scholar This validation included positivity and intensity threshold assessments, DNA quantification according to the nature of the biological sample, correlation comparisons between ddPCR and qPCR, and repeatability. The total droplet number was determined, and only wells containing >10,000 droplets were accepted for analysis. To discriminate positive droplets from negative droplets, a positivity threshold was set in terms of the fluorescence intensity and number of positive droplets. The intensity threshold was calculated as the sum of the average of the fluorescence intensity of all droplets from 75 negative samples and values 3 times the standard deviation. To fix the droplet positivity threshold for T. gondii detection, samples of bronchoalveolar fluid and blood negative by qPCR were replicated 8 times and analyzed by ddPCR using REP-529 and α-tubulin. The amount of DNA in nanograms was calculated in the test DNA extract submitted to ddPCR for various clinical samples, including AH, BAL, CSF, AF, and placenta, thanks to the direct measurement of DNA concentration in nanograms per microliter with a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) (Supplemental Table S1). A regression line was performed with serial 2-, 5-, and 10-fold dilutions of a DNA extract from a reference strain of T. gondii RH (type I). Each dilution was analyzed in triplicate in two independent experiments. The correlation was established between log (mean concentration in copies/5 μL of REP-529) obtained by ddPCR and the mean CT value obtained by qPCR (Supplemental Tables S2 and S3). The repeatability of ddPCR was determined for six different biological samples (AF, BAL, blood, and AH) and replicated eight times in a single experiment (Supplemental Table S4). Absolute quantification was performed using the α-tubulin target, a one-copy gene of 1672 bp located on chromosome XI of the T. gondii genome.23Nagel S.D. Boothroyd J.C. The alpha- and beta-tubulins of Toxoplasma gondii are encoded by single copy genes containing multiple introns.Mol Biochem Parasitol. 1988; 29: 261-273Crossref PubMed Scopus (127) Google Scholar New consensus primers and probes were designed, and a standard curve of the ddPCR signal targeting the α-tubulin gene was generated to assess the linearity. Standard curve was generated with serial 2.5- and 10-fold dilutions of BAL samples positive for Toxoplasma gondii (sample 3, type III strain, TgH29139A) (Table 1 and Supplemental Table S5). Comparisons were performed between T. gondii parasite loads measured by α-tubulin ddPCR absolute quantification and estimated by REP-529 qPCR relative quantification (Supplemental Table S6). The number of T. gondii parasites estimated by REP-529 qPCR was based on a standard curve of RH reference strain (Supplemental Table S7 and Supplemental Figure S1).Table 1Clinicoepidemiologic Settings of the Patients along with Quantification of Toxoplasma gondii by α-Tubulin ddPCR Absolute Quantification and by REP-529 qPCR Relative QuantificationPatientAge, yearsSexCountryKey clinical pointsPositive sample by qPCR and ddPCR REP-529Quantified sampleSample no.qPCR REP-529, mean CT valuesqPCR REP-529, mean copies/mL∗Toxoplasma quantifications are expressed as the mean copies per milliliter of DNA eluate.,†The mean Toxoplasma gondii copies per milliliter estimated by qPCR REP-529 were based on a standard curve of an RH reference strain (Supplemental Tables S6 and S7 and Supplemental Fig. S1).ddPCR α-tubulin, mean copies/mL)∗Toxoplasma quantifications are expressed as the mean copies per milliliter of DNA eluate.Genotype‡Number of amplified microsatellite markers is indicated into brackets.StrainA58MReunion IslandReactivation, pulmonary toxoplasmosis, immunosuppressed, renal transplant, deathBlood, BALBlood128.8985,5641490––Blood230.7026,565473––BAL323.492,700,63859,310Type III (n = 14/15)TgH29139AB53MFranceReactivation, pulmonary toxoplasmosis, immunosuppressed, liver transplant, deathBlood, BALBlood426.38422,2516250––BAL525.72645,69510,040Type III (n = 15/15)TgH29148AC49FFranceReactivation, cerebral toxoplasmosis, immunosuppressed, HIV, deathBlood, CSFBlood623.303,045,88429,667Type II (n = 15/15)TgH29147AD38FFranceCongenital toxoplasmosisAFAF728.09140,988880Type II (n = 10/15)TgH29140AE33FFranceCongenital toxoplasmosisAFAF827.30235,0152980Type II (n = 14/15)TgH29143AF32MGabonReactivation, pulmonary toxoplasmosis, immunosuppressed, renal transplantBlood, BALBlood928.38117,2511752––BAL1031.6314,556342Type III (n = 15/15)TgH29146AG31FCamerounReactivation, ocular toxoplasmosis, immunosuppressed, HIVAHAH1128.9084,095651Atypical (n = 11/15)TgH29141AH26FMaliCongenital toxoplasmosisAFAF1224.311,593,86222,277Atypical (Africa 1) (n = 15/15)TgH29142AI15MFranceNo data availableBloodBlood1332.0910,860170Not amplifiedTgH29144AJ27FDRCOcular toxoplasmosis, immunocompetent, panuveitis, hyalitisAHAH1430.3333,510292Not amplifiedTgH29149AK63FFranceImmunosuppressed, cerebral toxoplasmosis, hemopathy, deathBlood, CSFCSF1529.1272,911497Type II (n = 12/15)TgH29145AControlsAF1624.181,738,98821,423Type IRHAF1723.173,325,21154,983Type IRHMouse brain1828.2131,6769357Type IIME49F, female; M, male; AF, amniotic fluid; AH, aqueous humour; BAL, bronchoalveolar fluid; CSF, cerebrospinal fluid; CT, threshold cycle; ddPCR, droplet digital; DRC, Democratic Republic of Congo; qPCR, real-time quantitative PCR.∗ Toxoplasma quantifications are expressed as the mean copies per milliliter of DNA eluate.† The mean Toxoplasma gondii copies per milliliter estimated by qPCR REP-529 were based on a standard curve of an RH reference strain (Supplemental Tables S6 and S7 and Supplemental Fig. S1).‡ Number of amplified microsatellite markers is indicated into brackets. Open table in a new tab F, female; M, male; AF, amniotic fluid; AH, aqueous humour; BAL, bronchoalveolar fluid; CSF, cerebrospinal fluid; CT, threshold cycle; ddPCR, droplet digital; DRC, Democratic Republic of Congo; qPCR, real-time quantitative PCR. DNA samples from the patients and controls positive for T. gondii were genotyped at Limoges National Reference Centre Laboratory using 15 multilocus microsatellite markers distributed on 10 of 14 chromosomes, according to a previously published protocol.24Ajzenberg D. Collinet F. Mercier A. Vignoles P. Dardé M.L. Genotyping of Toxoplasma gondii isolates with 15 microsatellite markers in a single multiplex PCR assay.J Clin Microbiol. 2010; 48: 4641-4645Crossref PubMed Scopus (145) Google Scholar GraphPad Prism software version 9 (GraphPad Software, San Diego, CA) was used for the generation of curves, histograms, and box plots. For each correlation test, the equation of the simple linear regression is provided, as well as the linear determination coefficient (R2), to determine the quality of the prediction of the linear regression. Agreement and correlation between ddPCR quantification and cycle threshold values obtained by qPCR were evaluated by the Bland-Altman method comparison test.25Martin Bland J. Altman D.G. Statistical methods for assessing agreement between two methods of clinical measurement.Lancet. 1986; 327: 307-310Abstract Scopus (40229) Google Scholar In this study, the Bland-Altman represents, for each sample, the difference between the log (mean concentration in copies per milliliter of REP-529) obtained by ddPCR and the mean qPCR cycle threshold values, plotted against the mean of these two values. The clinical samples were deidentified before research and not linked to clinical records. Thus, the study does not fulfill criteria for human subjects research. At the time of sampling, none of the patients expressed opposition to the use of their samples for scientific purposes. The study is thus exempt from ethics committee approval, in accordance with French law 2004-806, dated August 9, 2004, on public health policy. The intensity threshold was set to 5160 fluorescence units for REP-529 and 2300 fluorescence units for α-tubulin. One droplet at most from one replicate of eight was observed above the selected intensity threshold in the two negative tested samples (Figure 1A). Therefore, samples with at least two droplets were considered positive. The amount of DNA varied greatly, depending on the type of biological sample (Figure 1B). Nevertheless, the amount of DNA did not impact parasite detection by ddPCR. A good correlation (R2 = 0.99) was observed between the ddPCR and real-time qPCR CT values using the REP-529 target and an RH strain (Figure 1C). The repeatability was high for the six tested samples, and the good precision was reflected by low CV (Figure 1D). A good correlation was observed between ddPCR and qPCR (R2 = 0.92) (Figure 2A), and the overall concordance between ddPCR and qPCR was 96.7% (n = 145/150). The Bland-Altman plot and linear regression showed very good agreement with no sample outside the ±1.96 SDs area and a high level of correlation (R2 = 0.97) (Figure 2B). Among the 150 samples of the study cohort, 35 tested positive for T. gondii, 110 tested negative, and 5 were discordant between the ddPCR and qPCR results. Positive samples by qPCR included blood (n = 13/100), AH (n = 10/20), AF (n = 6/12), BAL (n = 3/7), and CSF (n = 3/7). The four quality controls were concordant and valid. The five discordant results were positive only by ddPCR, and they were based on only two positive droplet signals. One of them was a CSF sample (patient B) (Table 1) that yielded one positive replicate by ddPCR, as confirmed in an independent experiment. Interestingly, for this patient, positive blood and BAL test results were recorded approximately 7 days later. The other discordant samples were more likely to correspond to false-positive results by ddPCR, with an AH sample obtained from a patient with negative serologic test results for T. gondii and three blood samples with only one positive replicate. The PCR primers for the α-tubulin gene amplified a 144-bp fragment and showed good specificity for T. gondii (Figure 3A). Indeed, all the negative samples using REP-529 also tested negative using α-tubulin. In addition, good linearity of the ddPCR signal was obtained using the α-tubulin newly designed target (R2 = 0.99) (Figure 3B). Among the 35 positive samples detected by ddPCR using REP-529, absolute quantification using α-tubulin was achievable for 15 samples (42.8%) from 11 patients. Three positive controls for the RH and ME49 reference strains were also successfully quantified. Quantification results by ddPCR along with clinical issues and T. gondii genotypes are summarized in Table 1. Coefficient of correlation between the α-tubulin ddPCR absolute quantification and REP-529 qPCR cycle threshold results estimated regardless of the genotype was R2 = 0.91 (Figure 3C). Compared with ddPCR absolute quantification, the parasite load was overestimated using the REP-529 qPCR relative quantification method (Figure 3D). For the same patient strain but different clinical samples, the ratio between the theoretical parasite load by qPCR and the measured parasite load by ddPCR was close, as shown for patients A (46, 56, and 57), B (68 and 64), and F (67 and 43). Between the T. gondii strains, the ratio showed greater variations within and between lineages. The largest variation was observed within the type II strains, with ratios from 14 to 160. This is the first study evaluating ddPCR for T. gondii detection and quantification in human clinical samples. A total of 150 samples were retrospectively studied. Good concordance and correlation were observed between the ddPCR and qPCR results for T. gondii detection using REP-529. Absolute quantification of T. gondii by ddPCR using an α-tubulin single-copy target was successful for 15 of 35 samples showing a parasite load ≥170 copies/mL of DNA eluate. We have thus shown that ddPCR is a robust method with good linearity and good precision for T. gondii detection and quantification. This result is in agreement with other studies also reporting good linearity and precision results for other pathogen detection.17Kuypers J. Jerome K.R. Applications of digital PCR for clinical microbiology.J Clin Microbiol. 2017; 55: 1621-1628Crossref PubMed Scopus (135) Google Scholar,19Lei S. Chen S. Zhong Q. Digital PCR for accurate quantification of pathogens: principles, applications, challenges and future prospects.Int J Biol Macromol. 2021; 184: 750-759Crossref PubMed Scopus (20) Google Scholar For T. gondii detection, ddPCR and qPCR shared a high performance, as indicated by the high level of agreement between the results, but ddPCR did not show clear superiority. Contrasting results were reported in terms of the higher accuracy of ddPCR compared with qPCR for parasitologic diagnosis. For example, a higher sensitivity of ddPCR compared with qPCR has been shown for all Plasmodium species detection of subpatent parasitemia samples in duplex ddPCR,26Srisutham S. Saralamba N. Malleret B. Rénia L. Dondorp A.M. Imwong M. Four human Plasmodium species quantification using droplet digital PCR.PLoS One. 2017; 12e0175771Crossref PubMed Scopus (45) Google Scholar whereas another study observed a higher sensitivity of ddPCR to diagnose Plasmodium falciparum but equal sensitivity for Plasmodium vivax.27Koepfli C. Nguitragool W. Hofmann N.E. Robinson L.J. Ome-Kaius M. Sattabongkot J. Felger I. Mueller I. Sensitive and accurate quantification of human malaria parasites using droplet digital PCR (ddPCR).Sci Rep. 2016; 6: 39183Crossref PubMed Scopus (73) Google Scholar This study presents a valuable assessment of the ddPCR method for T. gondii detection and quantification. The ddPCR results were compared with those of the qPCR reference method, which is currently used in the routine diagnosis of T. gondii. Both negative and positive samples for six different biological sample types that are commonly tested (blood, BAL, CSF, AF, placenta, and AH) were successfully evaluated. Among the positive samples, five different genotypes, including type I, type II, type III, and two atypical strains, were also successfully tested. Patients with T. gondii exhibited various typical clinical features, including reactivation, ocular toxoplasmosis, and congenital toxoplasmosis. Nevertheless, the study did not allow for the evaluation of the impact of absolute T. gondii quantification in the clinical management of T. gondii cases and in the prognosis of patients because of the low number of cases for each clinical entity. In addition, the quantification of parasite load using α-tubulin was achievable only for 43% of samples positive by qPCR REP-529 because of lower sensitivity of the single-copy target, require for absolute quantification. However, the lowest parasite load measured by α-tubulin ddPCR was 170 copies/mL of DNA eluate (ie, 0.17 copy/μL), which is a low limit of quantification, equivalent to that observed in the literature with other DNA targets. Although not previously reported for T. gondii using ddPCR, for P. falciparum protozoan parasite, the lowest measured parasite load was 0.7 copy/μL in mosquito midgut28Wang C.Y.T. McCarthy J.S. Stone W.J. Bousema T. Collins K.A. Bialasiewicz S. Assessing Plasmodium falciparum transmission in mosquito-feeding assays using quantitative PCR.Malar J. 2018; 17: 249Crossref PubMed Scopus (17) Google Scholar and approximately 1 copy/μL in human blood.27Koepfli C. Nguitragool W. Hofmann N.E. Robinson L.J. Ome-Kaius M. Sattabongkot J. Felger I. Mueller I. Sensitive and accurate quantification of human malaria parasites using droplet digital PCR (ddPCR).Sci Rep. 2016; 6: 39183Crossref PubMed Scopus (73) Google Scholar For another protozoan parasite, Trypanosoma cruzi, it was 5 copies/μL,29Ramírez J.D. Herrera G. Hernández C. Cruz-Saavedra L. Muñoz M. Flórez C. Butcher R. Evaluation of the analytical and diagnostic performance of a digital droplet polymerase chain reaction (ddPCR) assay to detect Trypanosoma cruzi DNA in blood samples.PLoS Negl Trop Dis. 2018; 12e0007063Crossref Scopus (15) Google Scholar whereas for Aspergillus fumigatus filamentous fungus, it was 0.2 and 0.3 copy/μL.30Alanio A. Sturny-Leclère A. Benabou M. Guigue N. Bretagne S. Variation in copy number of the 28S rDNA of Aspergillus fumigatus measured by droplet digital PCR and analog quantitative real-time PCR.J Microbiol Methods. 2016; 127: 160-163Crossref PubMed Scopus (20) Google Scholar Interestingly, the limit of quantification of α-tubulin ddPCR was comparable to the limit of qPCR amplification of microsatellite markers of T. gondii, as a complete genotype could be obtained only above 292 copies/mL. Diversity of strain genotypes did not appear to influence the accuracy of REP-529 qPCR detection and α-tubulin ddPCR quantification, as revealed by the high level of correlation between the ddPCR absolute quantification and REP-529 qPCR CT. A previous study also showed that the genotype did not impact the detection performance using the REP-529 target.31Pomares C. Estran R. Press C.J. Bera A. Ramirez R. Montoya J.G. Gangneux F.R. Is real-time PCR targeting rep 529 suitable for diagnosis of toxoplasmosis in patients infected with non-type II strains in North America?.J Clin Microbiol. 2020; 58e01223-19Crossref PubMed Scopus (8) Google Scholar However, we demonstrated that the genotype impacted the quantification performance using REP-529 qPCR, probably because of the variable number of REP-529 copies. Nevertheless, the absolute number of copies of REP-529 per parasite strain could not be directly provided by absolute ddPCR quantification in this study because the target was multicopy. To assess the number of REP-529 copies per parasite using ddPCR, a prior restriction enzyme digestion is needed to separate each copy of REP-529 and to ensure the inclusion of a single copy of REP-529 by ddPCR reaction. In such conditions, the ratio between REP-529 gene concentration and α-tubulin gene concentration could provide the number of REP-529 copies per parasite. Using our new absolute quantification assay targeting a specific region of α-tubulin of T. gondii, we demonstrated that ddPCR may be used if the qPCR REP-529 results show an approximately amplification CT <32. From our perspective, it would be preferable to use qPCR as a screening tool for T. gondii and to perform ddPCR only for absolute quantification purposes of positive qPCR samples because of the higher complexity and lower throughput of ddPCR than qPCR. Absolute quantification for T. gondii could be useful for specific clinical scenarios, such as examining pathogen clearance after starting antitoxoplasma therapy in a severely immunocompromised patient who seems not to be responding adequately to treatment. Indeed, several concomitant syndromes, such as immune reconstitution inflammatory syndrome or the presence of another opportunistic infection, can be masking or misinterpreted as toxoplasma treatment failure. The assessment of parasitic burden could also be useful to evaluate the prognosis of the disease in the case of congenital toxoplasmosis and for transplant and hematologic immunocompromised hosts by providing a parasite load threshold correlating with prognosis, but further studies are needed. Finally, ddPCR could prove useful for laboratory practice for the precise quantification of standards. It also responds to the need for laboratory standardization of the quantification of T. gondii. This would greatly enhance comparability of parasite loads from different laboratories and facilitate multicentric studies. ddPCR is an emerging method for the diagnosis and monitoring of microbial infections. This proof of concept shows the high performance of ddPCR for Toxoplasma gondii detection and absolute quantification for a large variety of clinical samples. We have shown that ddPCR offers a more accurate T. gondii quantification compared with qPCR. Thereby, molecular tools, such as ddPCR, open the door to new clinical applications by improving the therapeutic monitoring of toxoplasmosis and the molecular assessment of parasitic burden. To better assess the clinical impact of T. gondii absolute quantification, a multicentric, prospective study should now be performed, with samples from specific clinical cohorts, such as pregnant women and immunosuppressed patients after graft transplant in hematology or after solid organ transplant.