Hélène Yera

Congenital Toxoplasmosis and Reinfection during Pregnancy: Case Report, Strain Characterization, Experimental Model of Reinfection, and Review

We present a case of disseminated congenital toxoplasmosis in a newborn born to a mother who had been immunized against toxoplasmosis before conception. The mother was reinfected, likely by ingestion of imported raw horse meat during pregnancy. This clinical presentation is exceptional in France and raised the possibility of infection by a highly virulent Toxoplasma strain. The strain responsible was isolated from the peripheral blood of the newborn, and when genotyped with microsatellite markers, it exhibited an atypical genotype, one which is very uncommon in Europe but had been described in South America. We tested the hypothesis of a reinfection with a different genotype by using an experimental mouse model, which confirmed that acquired immunity against European Toxoplasma strains may not protect against reinfection by atypical strains acquired during travel outside Europe or by eating imported meat.

Contributions of Immunoblotting, Real-Time PCR, and the Goldmann-Witmer Coefficient to Diagnosis of Atypical Toxoplasmic Retinochoroiditis

ABSTRACT Ocular toxoplasmosis is a major cause of posterior uveitis worldwide. The diagnosis is based mainly on ophthalmological examination. Biological diagnosis is necessary in atypical cases, and this requires aqueous humor sampling by anterior chamber paracentesis. We evaluated real-time PCR targeting the Toxoplasma gondii 529-bp repeat element, the Goldmann-Witmer coefficient (GWC), and immunoblotting for the diagnosis of toxoplasmic retinochoroiditis in 54 patients with atypical uveitis. The results of these biological tests, applied to paired aqueous humor-serum samples, were compared to the clinical findings. Combining either PCR or the GWC with immunoblotting increased the sensitivity to 73% or 70%, respectively. Together, PCR and the GWC had 80% sensitivity. If feasible, sensitivity can be increased by combining the three methods (85% sensitivity). The interval between symptom onset and anterior chamber paracentesis strongly influenced the detection of specific intraocular antibody synthesis. The sensitivity of the GWC increased from 45% to 56% when sampling was performed 10 days after symptom onset, and that of immunoblotting increased from 53% to 72% when puncture was performed 30 days after symptom onset. PCR analysis of aqueous humor samples detected toxoplasmic DNA in 55% of patients. In contrast to the results of immunoblotting and the GWC, the results of PCR were not influenced by the interval between symptom onset and paracentesis. PCR was more informative than the GWC and immunoblotting for immunocompromised patients. Acute necrotizing retinal lesions were significantly larger in PCR-positive patients, with a mean of 3.5 optic disc diameters, than in PCR-negative patients, with a mean of 1.5 optic disc diameters.

Development and Evaluation of a Western Blot Kit for Diagnosis of Human Trichinellosis

ABSTRACT We evaluated industrially prepared Western blot strips designed to avoid the cross-reactions observed with indirect immunofluorescence and enzyme-linked immunosorbent assays used for the serodiagnosis of trichinellosis. The antigen preparations were crude extracts of Trichinella spiralis. The Western blot profile characteristic of trichinellosis was characterized by comparing 60 sera from patients infected by Trichinella to 11 sera from healthy subjects, 51 sera from patients with other proven parasitic diseases (cysticercosis, schistosomiasis, strongyloidosis, fascioliasis, toxocariasis, liver amebiasis, anisakiasis, filariasis, toxoplasmosis, hydatidosis, or malaria), and 23 sera from patients with autoantibodies. Specific 43- to 44-kDa and 64-kDa bands were obtained with all of the sera from 51 patients with acute trichinellosis, in 4 out of 9 patients at the early stages of the disease, and in only 1 control patient, who had suspected anisakiasis and in whom trichinellosis could not be ruled out by muscle biopsy.

Comparison of PCR, microscopic examination and culture for the early diagnosis and characterization of Acanthamoeba isolates from ocular infections

In the study presented here, PCR, microscopic examination and culture of corneal samples were compared as methods of confirming the clinical diagnosis of Acanthamoeba keratitis, a serious ocular infection that is difficult to diagnose and threatens eyesight. The three methods were applied to isolates obtained from 513 patients with clinical signs or risk factors suggesting Acanthamoeba infection. Acanthamoeba keratitis was diagnosed in 12 of these patients. Combined PCR assays were more sensitive (94%) than either microscopic examination (33%) or culture (7%). The Acanthamoeba isolates were characterized using DNA sequence analysis of the nuclear small-subunit rRNA gene, and T4 was the predominant genotype found.

Multicentric Comparative Analytical Performance Study for Molecular Detection of Low Amounts of Toxoplasma gondii from Simulated Specimens

ABSTRACT Although screening for maternal toxoplasmic seroconversion during pregnancy is based on immunodiagnostic assays, the diagnosis of clinically relevant toxoplasmosis greatly relies upon molecular methods. A problem is that this molecular diagnosis is subject to variation of performances, mainly due to a large diversity of PCR methods and primers and the lack of standardization. The present multicentric prospective study, involving eight laboratories proficient in the molecular prenatal diagnosis of toxoplasmosis, was a first step toward the harmonization of this diagnosis among university hospitals in France. Its aim was to compare the analytical performances of different PCR protocols used for Toxoplasma detection. Each center extracted the same concentrated Toxoplasma gondii suspension and tested serial dilutions of the DNA using its own assays. Differences in analytical sensitivities were observed between assays, particularly at low parasite concentrations (≤2 T. gondii genomes per reaction tube), with “performance scores” differing by a 20-fold factor among laboratories. Our data stress the fact that differences do exist in the performances of molecular assays in spite of expertise in the matter; we propose that laboratories work toward a detection threshold defined for a best sensitivity of this diagnosis. Moreover, on the one hand, intralaboratory comparisons confirmed previous studies showing that rep529 is a more adequate DNA target for this diagnosis than the widely used B1 gene. But, on the other hand, interlaboratory comparisons showed differences that appear independent of the target, primers, or technology and that hence rely essentially on proficiency and care in the optimization of PCR conditions.

Multicenter Comparative Evaluation of Five Commercial Methods for Toxoplasma DNA Extraction from Amniotic Fluid

ABSTRACT Over the past few years, a number of new nucleic acid extraction methods and extraction platforms using chemistry combined with magnetic or silica particles have been developed, in combination with instruments to facilitate the extraction procedure. The objective of the present study was to investigate the suitability of these automated methods for the isolation of Toxoplasma gondii DNA from amniotic fluid (AF). Therefore, three automated procedures were compared to two commercialized manual extraction methods. The MagNA Pure Compact (Roche), BioRobot EZ1 (Qiagen), and easyMAG (bioMérieux) automated procedures were compared to two manual DNA extraction kits, the QIAamp DNA minikit (Qiagen) and the High Pure PCR template preparation kit (Roche). Evaluation was carried out with two specific Toxoplasma PCRs (targeting the 529-bp repeat element), inhibitor search PCRs, and human beta-globin PCRs. The samples each consisted of 4 ml of AF with or without a calibrated Toxoplasma gondii RH strain suspension (0, 1, 2.5, 5, and 25 tachyzoites/ml). All PCR assays were laboratory-developed real-time PCR assays, using either TaqMan or fluorescent resonance energy transfer probes. A total of 1,178 PCRs were performed, including 978 Toxoplasma PCRs. The automated and manual methods were similar in sensitivity for DNA extraction from T. gondii at the highest concentration (25 Toxoplasma gondii cells/ml). However, our results showed that the DNA extraction procedures led to variable efficacy in isolating low concentrations of tachyzoites in AF samples (<5 Toxoplasma gondii cells/ml), a difference that might have repercussions since low parasite concentrations in AF exist and can lead to congenital toxoplasmosis.

Molecular Diagnosis of Toxoplasmosis in Immunocompromised Patients: a 3-Year Multicenter Retrospective Study

ABSTRACT 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.

The genotypic characterisation of Acanthamoeba isolates from human ocular samples

Background: We characterised 37 amoebae cultured from corneal scrapings, contact lenses or lens case solutions of patients with suspected Acanthamoeba keratitis. Methods: The isolates were identified by their morphology and by PCR targeting the Acanthamoeba nuclear small-subunit rRNA gene. Acanthamoeba isolates were genotyped by DNA sequence analysis. Results: The 37 isolates comprised 35 Acanthamoeba, one Hartmannella and one Vahlkampfia. Ten Acanthamoeba isolates were shown to be responsible for keratitis. Conclusion: Genotype T4 was the only Acanthamoeba genotype responsible for keratitis in this study, and represented 79% of non-pathogenic isolates.

Pneumocystosis: a network survey in the Paris area 2003–2008

The aims of this network group were to collect epidemiological data of PcP cases in 14 hospitals in the Paris area and to determine the Di-Hydro Pteroate Synthase (DHPS) genotypes, genetic markers for possible sulfamide resistance. From January 1, 2003 to December 31, 2008, 993 (mean 166/year) PcP cases have been reported. Sixty-five percent of patients were HIV-positive. The median count of CD4 lymphocytes was 32/mm3 (30 in HIV-positive patients, 152 in HIV-negative patients). In HIV-positive patients, PcP revealed the HIV infection in 39%. Among 304 PcP occurring in HIV known infected patients, no prophylaxis was prescribed for 64%; cotrimoxazole prophylaxis had been prescribed to 47 patients but only one of them had the right compliance. In HIV-negative patients (264), corticosteroids were prescribed in 59% and cytotoxic chemotherapies in 34%; 78% did not receive prophylaxis. One hundred sixty nine tumoral pathologies and 116 transplantations were notified. The mortality rate was 16% at day 14 (13% in HIV-positive patients, 26% in HIV-negative patients). Mutations in DHPS genes were detected in 18.5% of samples; 12.5% of patients were infected with several strains. The total annual number of cases has been stable for five years but the proportion of HIV-negative patients increased from 25% to 43%.

Contribution of molecular diagnosis to congenital toxoplasmosis.

We evaluated the performance of three real-time polymerase chain reaction (PCR) assays on 73 samples from mothers and children with congenital toxoplasmosis. PCR assays had significantly higher sensitivity in prenatal period than in birth period when targeting the 529-bp repeat element (81.3% versus 36.0%) or the B1 gene (64.6% versus 20.0%).