Assessment of Droplet Digital PCR for the Detection and Absolute Quantification of Toxoplasma gondii: A Comparative Retrospective Study.

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.

Interpretation of very low avidity indices acquired with the Liaison XL Toxo IgG avidity assay in dating toxoplasmosis infection.

Congenital toxoplasmosis is an important cause of complications in pregnancy. Toxoplasmosis is often asymptomatic and thus serological tests are usually performed to screen for it. A first serum which exhibit both IgG and IgM may be due to nascent toxoplasmosis seroconversion, non-specific IgM reaction, or residual IgM. The IgG avidity test has been proposed to identify latent infections. A high index excludes recent toxoplasmosis whereas an intermediate or low index only suggests a recent infection, the caveats being that some people with latent Toxoplasma gondii infection show IgG with low or intermediate avidity. In this study, we investigated the ability of the Liaison XL Toxo IgG avidity (DiaSorin, Saluggia, Italy) assay to confirm recent infection when IgG avidity index is very low (≤ 0.1). Four thousand two hundred ninety-seven sera exhibiting both IgG and IgM were included and avidity was performed on the Liaison device according to the manufacturer's recommendations. One hundred twenty-six sera on the 297 sera which exhibited very low IgG avidity indices (≤ 0.1) could be exploited: 97% of sera with IgG avidity indices < 0.05 actually corresponded to recent infection (less than 3 months). A similar but less pronounced trend was observed for the sera exhibiting indices between 0.05 and 0.1 (69% corresponded to recent infections). The IgG avidity index data we obtained with the Liaison XL Toxo device are similar to those obtained with other devices. This body of consistent results underlines the interest of very low IgG avidity indices as a sign of probable recent toxoplasmosis.

Aspergillus PCR in Bronchoalveolar Lavage Fluid for the Diagnosis and Prognosis of Aspergillosis in Patients With Hematological and Non-hematological Conditions.

Objectives: We evaluated the usefulness of an Aspergillus fumigatus quantitative PCR assay performed in bronchoalveolar lavage fluid (BAL) for the diagnosis and prognosis of both invasive and non-invasive aspergillosis. Methods: This 4-year retrospective study involved 613 at-risk patients who had either hematological disorders or other immunosuppressive conditions, notably solid organ transplants. Thirty-five patients had proven/probable aspergillosis and thirteen had chronic non-invasive aspergillosis. We compared PCR, galactomannan index and mycological analysis of BAL. Results: For invasive aspergillosis (IA), PCR performed in BAL yielded 88.6% sensitivity and 95.5% specificity. Comparatively, galactomannan index and mycological examination yielded only 56.3 and 63.6% sensitivity and 97.6 and 94.5% specificity, respectively. Considering the 13 chronic aspergillosis cases, PCR, galactomannan index and mycological examination yielded 76.9, 15.4, and 84.6% sensitivity and 92.2, 94.9, and 93% specificity, respectively. Fungal load in BAL evaluated by PCR was able to discriminate between aspergillosis and contamination, but not between invasive and non-invasive forms. Finally, fungal load was predictive of 90-day mortality, with 23.1% mortality for patients with less than 500 copies/mL versus 68.4% for patients above that cut-off (p < 0.05). Conclusion: Our results indicate that Aspergillus PCR in BAL is of particular interest for both the diagnosis and the prognosis of IA. It is likewise an interesting tool for the diagnosis of non-invasive forms.

The impact of lowering the cut-off value on the sensitivity of the Platelia Elisa IgG (Bio-Rad) test for toxoplasmosis diagnosis.

Determining specific immune status against Toxoplasma gondii is essential for assessing the risk of reactivation in immunocompromised patients or defining serological monitoring and appropriate prophylactic measures during pregnancy. In France, toxoplasmosis serological screening requires systematic testing for IgM and IgG antibodies. The Platelia Toxo IgG and IgM test (Bio-Rad) is one of the most widely used tests for anti-toxoplasmic antibody detection. We performed a study on 384 sera, including 123 IgG negative (<6 IU/mL) and 261 IgG equivocal (6-9 IU/mL) sera tested with Platelia Toxo IgG and collected during routine screening at Pitié-Salpêtrière Hospital, Paris, France to determine the best-performing IgG titer cut-off value. Out of these 383 sera, 298 were IgM negative by Platelia Toxo IgM and 86 were IgM positive. All sera were also tested against Toxo IgG II LD BIO western blot test as confirmation. Our results indicated that an IgG titer cut-off value of ≥4.4 IU/mL for the Platelia Toxo IgG met the definition of positivity, a value significantly lower than that indicated by the manufacturers. In the presence of IgM antibodies, the IgG titer cut-off decreased significantly to a value ≥0.2 IU/mL. This latter cut-off also allowed adequate diagnosis of proven toxoplasmosis seroconversion in 76.7% of cases (33/43). Our findings may improve toxoplasmosis care by reducing therapeutic intervention time and eliminating the need for further serological monitoring.

Molecular diagnosis of toxoplasmosis: value of the buffy coat for the detection of circulating Toxoplasma gondii.

Early detection of Toxoplasma tachyzoites circulating in blood using PCR is recommended for immunosuppressed patients at high risk for disseminated toxoplasmosis. Using a toxoplasmosis mouse model, we show that the sensitivity of detection is higher using buffy coat isolated from a large blood volume than using whole blood for this molecular monitoring.

Molecular diagnosis of toxoplasmosis in immunocompromised patients: a 3-year multicenter retrospective study.

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.

Multicentric comparative assessment of the bio-evolution Toxoplasma gondii detection kit with eight laboratory-developed PCR assays for molecular diagnosis of congenital toxoplasmosis.

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.

Freezing and storage at -20 °C provides adequate preservation of Toxoplasma gondii DNA for retrospective molecular analysis.

Nucleic acid-based testing has become crucial for toxoplasmosis diagnosis. For retrospective (forensic or scientific) studies, optimal methods must be employed for DNA long-term storage. We compared Toxoplasma gondii detection before and after DNA storage using real-time PCR. No significant differences were found depending on duration or storage conditions at -20 °C or -80 °C.

Characterization and multicentric validation of a common standard for Toxoplasma gondii detection using nucleic acid amplification assays.

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.

Amebic liver abscess diagnosed by polymerase chain reaction in 14 returning travelers.

Amebic liver abscesses (ALA) are not commonly described in travelers. The ALA diagnosis is usually based on serology and Entamoeba histolytica polymerase chain reaction (PCR) is a new tool. We retrospectively reviewed all ALA cases diagnosed by PCR on the liver abscess pus aspirate of patients admitted in 4 teaching hospitals in Paris, France between 2007 and 2011. Fourteen cases (10 male, median age 48 years) were included. The median lag time between return and onset of symptoms was 23 days among 10 patients (interquartile range [IQ] 18­24) whereas the remaining patients had travelled over 2 years ago.All patients had an elevated C-reactive protein level, and 11 had leukocytosis. The ALA was multiple in five patients, localized in the right lobe in 12, and higher than 5 cm in 11. Serology was initially negative in one patient, whereas PCR was positive. There was bacterial co-infection in one patient. The outcome was good. Liver puncture allows a rapid diagnosis of ALA with PCR and helps identify the association with a bacterial dual infection [corrected]..