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.

Toxoplasma gondii infection in sheep from Romania.

BACKGROUND: Toxoplasmosis is a widespread zoonosis caused by the intracellular protozoan parasite Toxoplasma gondii. Limited epidemiological information is available about the prevalence of T. gondii in sheep in Romania, and a high incidence would have implications for both the economy and public health. To our knowledge, no studies are available about the T. gondii strains circulating in lambs. The objective of this study was to assess the prevalence of T. gondii in sheep (serology), lambs (serology, bioassay, PCR) and sheep abortions (PCR) in Romania. Moreover, the study aimed to perform the genetic characterization of T. gondii isolates from lambs. METHODS: Serum samples collected from 2650 sheep (2067 adults and 583 lambs) were tested for anti-T. gondii antibodies (IgG) using a commercial ELISA kit. Likewise, 328 pairs of diaphragmatic muscle-serum samples were collected from lambs aged between 2 and 4 months. Lamb serum samples were analyzed using MAT for anti-T. gondii antibody detection. The diaphragm tissue samples from MAT-positive lambs (at a dilution ≥ 1:25) were bioassayed in mice. The T. gondii strains were genotyped using 15 microsatellites markers. Additionally, brain and heart samples from 76 sheep abortions were analyzed for T. gondii DNA by polymerase chain reaction (PCR) targeting the 529-bp repeat region (REP529). RESULTS: The results showed that more than half of the tested sheep were T. gondii seropositive (53.5%). The seroprevalence was significantly higher in adults (61.1%) than in lambs (26.4%). The seroprevalence of T. gondii infection in slaughtered lambs, by MAT, was 37.5% (123/328). There were bioassayed in mice 56 diaphragmatic tissues from 123 seropositive lambs. Toxoplasma gondii strains were isolated from 18 (32.1%) lambs intended for human consumption. All T. gondii strains were confirmed by PCR. Six strains were genotyped using 15 microsatellite markers and belonged to genotype II. Toxoplasma gondii DNA was detected in 11.8% (9/76) of sheep abortions. CONCLUSIONS: The present study showed the presence of T. gondii in sheep in all the regions considered in the study. The high prevalence of T. gondii infection in sheep and lambs, demonstrated by serology, molecular analysis and bioassay, highlighted that there is an important risk of human infection in consuming raw or undercooked sheep/lamb meat.

Toxoplasmosis in patients with an autoimmune disease and immunosuppressive agents: A multicenter study and literature review.

BACKGROUND: Cases of Toxoplasma reactivation or more severe primary infection have been reported in patients receiving immunosuppressive (IS) treatment for autoimmune diseases (AID). The purpose of this study was to describe features of toxoplasmosis occurring in patients with AID treated by IS therapy, excluded HIV-positive and transplant patients. METHODS: A multicenter descriptive study was conducted using data from the French National Reference Center for Toxoplasmosis (NRCT) that received DNA extracts or strains isolated from patients, associated with clinical data. Other cases were retrieved through a questionnaire sent to all French parasitology and internal medicine departments. Furthermore, a systematic literature review was conducted. RESULTS: 61 cases were collected: 25 retrieved by the NRCT and by a call for observations and 36 from a literature review. Half of the cases were attributed to reactivation (50.9%), and most of cases (49.2%) were cerebral toxoplasmosis. The most common associated AID were rheumatoid arthritis (28%) and most frequent treatments were antimetabolites (44.3%). Corticosteroids were involved in 60.7% of cases. Patients had a favorable outcome (50.8%) but nine did not survive. For 12 cases, a successful Toxoplasma strain characterization suggested the possible role of this parasitic factor in ocular cases. CONCLUSION: Although this remains a rare condition, clinicians should be aware for the management of patients and for the choice of IS treatment.

Toxoplasmosis Outbreak Associated With Toxoplasma gondii-Contaminated Venison-High Attack Rate, Unusual Clinical Presentation, and Atypical Genotype.

BACKGROUND: During 2017, in response to a physician's report, the Wisconsin Department of Health Services, Division of Public Health, began investigating an outbreak of febrile illness among attendees of a retreat where never frozen, intentionally undercooked, locally harvested venison was served. Preliminary testing tentatively identified the illness as toxoplasmosis. METHODS: Confirmatory human serology panels and testing of the venison to confirm and categorize the presence and type of Toxoplasma gondii were completed by French and American national reference laboratories. All 12 retreat attendees were interviewed; medical records were reviewed. RESULTS: All attendees were male; median age was 51 years (range: 22-75). After a median incubation period of 7 days, 9 (82%) of 11 exposed persons experienced illness lasting a median of 12 days. All 9 sought outpatient healthcare for symptoms including fever, chills, sweats, and headache (100%) and ocular disturbances (33%). Testing confirmed the illness as toxoplasmosis and venison as the infection source. Multiple laboratory results were atypical for toxoplasmosis, including transaminitis (86%), lymphocytopenia (88%), thrombocytopenia (38%), and leukopenia (63%). One exposed but asymptomatic person was seronegative; the other had immunity from prior infection. The T. gondii strain was identified as closely related to an atypical genotype (haplogroup 12, polymerase chain reaction restriction fragment length polymorphism genotype 5) common in North American wildlife but with previously uncharacterized human clinical manifestations. CONCLUSIONS: The T. gondii strain contaminating the venison might explain the unusual clinical presentations. In North America, clinicians and venison consumers should be aware of risk for severe or unusual presentations of acute toxoplasmosis after consuming undercooked game meat.

A case of congenital toxoplasmosis-associated miscarriage with maternal infection four months prior to conception.

BACKGROUND: We report a case of fatal congenital toxoplasmosis with maternal infection dated four months before pregnancy in the absence of any specific immunosuppressive condition. CASE: Ms. D. experienced submaxillary lymphadenitis in February 2018. The medical workup performed revealed an acute T. gondii infection. She became pregnant in June 2018 while she still had adenopathy. The second obstetrical ultrasound, performed at 16 weeks of pregnancy, revealed a fetal death. The research for T. gondii by PCR was positive in the products of conception. CONCLUSION: Diagnosis of toxoplasmosis should be discussed in case of miscarriage with lymphadenitis. As lymph nodes in T. gondii infection could be responsible for iterative release of parasites and fetal death, symptomatic toxoplasmosis should be treated in women of childbearing age.

First isolation and molecular characterization of Toxoplasma gondii strains from human congenital toxoplasmosis cases in Monastir, Tunisia.

Toxoplasma gondii is a protozoon parasite that can cause severe clinical problems such as congenital toxoplasmosis. The distribution of T. gondii genotypes varies from one geographic area to another. So far, little is known about the parasite genotypes in Tunisia, North Africa. The present study aimed isolating and genotyping T. gondii from the amniotic fluid (AF) and placenta of pregnant women in Monastir, Tunisia. Amniotic fluid and/or placenta from 80 women who acquired toxoplasma infection during pregnancy were tested by PCR and/or mouse bioassay. Genotyping of T. gondii isolates from these samples was performed with 15 microsatellite markers. Four viable T. gondii strains were isolated from either the AF or placenta of four women. Specifically, strains TUN001-MON1 and TUN002-MON2 were isolated from both the AF and placenta, TUN003-AHA from only the placenta, and TUN004-NEL from only the AF. The four viable strains were not virulent for mice. Genotyping revealed that the four strains were type II strains. This is the first report on isolation and genotyping of T. gondii from AF human samples in Tunisia. Further studies focused on T. gondii genotyping on a larger number of human cases and on animals in Tunisia are needed to improve the knowledge and epidemiology of toxoplasmosis.

Evaluation of a panel of molecular markers for the diagnosis of malignant serous effusions.

PURPOSE: Our main goal was to evaluate a panel of molecular markers for the detection of cancer cells in serous effusions and to determine their value as an adjunctive reverse transcription-PCR (RT-PCR) test to cytologic examination. EXPERIMENTAL DESIGN: One hundred fourteen serous effusions from 71 patients with tumors and 43 patients with benign diseases were subjected to RT-PCR for expression of carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (Ep-CAM), E-cadherin, mammaglobin, mucin 1 (MUC1) isoforms MUC1/REP, MUC1/Y, and MUC1/Z, calretinin, and Wilms' tumor 1 susceptibility gene. RESULTS: CEA, Ep-CAM, E-cadherin, and mammaglobin were specifically expressed in malignant effusions. The sensitivity of RT-PCR in cytologically negative malignant effusions was 63.1% combining CEA and Ep-CAM (with 100% specificity) and reached 78.9% adding MUC1/Y or MUC1/Z (with 93% specificity). In the whole population of effusions, the combination of cytology with RT-PCR of CEA and Ep-CAM yielded a 90.1% sensitivity, a specificity and a positive predictive value of 100%, and a 86% negative predictive value for malignancy. Adding MUC1/Y or MUC1/Z to the panel, the sensitivity was 94.5% with 93% specificity, 95.7% PPV, and 90.9% negative predictive value. Moreover, CEA and mammaglobin were specifically expressed in epithelial malignancies, and mammaglobin was mainly expressed in effusions from breast carcinoma (97.3% of specificity). CONCLUSIONS: A combination of cytology and RT-PCR analysis of CEA and Ep-CAM significantly improved the detection sensitivity of tumor cells in serous effusions. RT-PCR analysis of CEA, Ep-CAM, and mammaglobin in serous effusions could be a beneficial adjunct to cytology for the diagnosis of malignancy.

Cadherin-6 gene expression in conventional renal cell carcinoma: a useful marker to detect circulating tumor cells.

BACKGROUND: Dissemination of cancer cells into the circulation is an essential step in the development of a metastasis. Detection of circulating cancer cells may improve the monitoring methods for cancer patients. However, the detection of circulating renal cancer cells is mainly hampered by the lack of markers available for renal cell carcinoma (RCC). In this study, we evaluated cadherin-6 mRNA as a new molecular marker for the detection of circulating renal cancer cells. MATERIALS AND METHODS: Forty-six blood samples of conventional RCCs were included. A standard protocol of RT-PCR, assisted by computer densitometric analysis to establish a cut-off, was performed to examine cadherin-6 mRNA expression by using specific primers. A renal cancer cell line, SKRC-59 and forty tumor biopsies from conventional RCCs were used as positive controls. Twenty-five blood samples from non-RCC patients were also analyzed. RESULTS: Cadherin-6 mRNA could be detected in 38140 (95%) conventional RCC specimens. Cadherin-6 mRNA was positive in 21/46 (45.7%) blood samples of RCC patients, while no positivity was found in non-RCC blood samples. Among the localized RCCs, 14/35 (40.0%) blood samples were positive while 7/11 (63.6%) were positive among the blood samples from metastatic RCCs. CONCLUSION: Our data indicate that cadherin-6 gene is frequently expressed in conventional RCCs. Cadherin-6 is a useful molecular marker to detect the circulating cancer cells disseminated from conventional RCC.

Rapid and sensitive detection of messenger RNA expression for molecular differential diagnosis of renal cell carcinoma.

PURPOSE: The aim of this study was to develop a practical technique to detect mRNA expression and to validate a panel of mRNA markers for molecular differential diagnosis of renal cell carcinoma (RCC). EXPERIMENTAL DESIGN: The renal cancer cell line SKRC-52 was used to set up the technique, which consisted of column extraction of RNA and one-step reverse transcription-PCR. We validated a panel of gene markers, including MN/CA9, cadherin-6, vimentin, mucin1, and parvalbumin, and studied 50 renal tumors (30 conventional, 9 papillary, and 5 chromophobe RCCs and 6 oncocytomas), 10 normal tissues, and 10 normal blood samples. We mimicked fine needle aspiration (FNA) biopsy in 10 kidneys with conventional RCC and applied this technique to 10 preoperative FNA samples from imaging-indeterminate renal tumors. RESULTS: The technique could detect as few as 10 SKRC-52 cells with MN/CA9 as mRNA marker and was less time consuming and labor intensive. MN/CA9 was a sensitive and rather specific gene marker for conventional RCC. Cadherin-6 gene expression was a sensitive marker for conventional and papillary RCC. Vimentin was highly specific for conventional RCC. Mucin1 mRNA was sensitive for papillary and chromophobe RCC and oncocytoma. Parvalbumin mRNA was a sensitive and highly specific marker for both chromophobe RCC and oncocytoma. Thus, these mRNA markers represent the biomarker genes for the subtypes of renal tumors. Finally, we successfully applied the technique to FNA specimens. Five preoperative FNA samples were MN/CA9 gene positive, suggesting a RCC, whereas the routine cytology was positive in only three cases. CONCLUSIONS: A rapid and sensitive assay of mRNA markers was developed for molecular differential diagnosis of RCC. This molecular assay can be used as a powerful ancillary to surgical pathological diagnosis and cytological diagnosis of RCC.

The in vivo DNA aneuploidization during expansion of conventional renal cell carcinoma.

Although numerous studies on the prognostic value of DNA aneuploidy in RCC have been reported, the in vivo DNA aneuploidization during RCC expansion has not been revealed. The present study was undertaken to observe the DNA aneuploidization during RCC expansion. We studied prospectively 67 consecutive conventional RCCs. The ploidy status was determined by analyzing five fresh tumor tissues from different areas by flow cytometry. The diploid, heterogeneous aneuploid tumors and homogeneous aneuploid tumors could be detected, respectively, in 44.8%, 23.9% and 31.3% of cases. The diploid tumors decreased significantly and aneuploid tumors increased significantly as the tumor expanded. The similar DNA content distribution was found between the heterogeneous aneuploid tumors and homogeneous aneuploid tumors. The hypertriploid clone was the most frequent in aneuploid tumors. The tumors of multiple aneuploid clones (16.4%) were mainly found in large-sized tumors. These results suggested that some RCCs underwent DNA aneuploidization during the tumor expansion and that a major route of aneuploidiztion (hypertriploidization) and several pathways existed. Our results also supported the idea that the progressive chromosomal instability was associated with continued tumor growth of RCC. The molecular mechanism and the clinical significance of aneuploidy phenotypes need to be further investigated.