Sophie Cassaing

Comparison between Two Amplification Sets for Molecular Diagnosis of Toxoplasmosis by Real-Time PCR

ABSTRACT PCR is now commonly applied to the diagnosis of toxoplasmosis. Although several methods are available, comparative studies are few, making it difficult to compare the performance of each technique. We compared the sensitivities of two real-time PCR assays through a prospective study on fetuses, neonates, and immunocompromised patients and on the ocular diagnosis of toxoplasmosis. The first system targeted the widely used B1 gene (GenBank accession number AF179871) while the second (RE) targeted a more recently described sequence repeated roughly 200 to 300 times (GenBank accession number AF146527). We demonstrated that molecular diagnosis requires the duplication of PCR assays, especially with the B1 system, as only one PCR was positive in 33.3% of cases. Our study showed that the RE target was more sensitive for all biological samples (amniotic fluid, placenta, aqueous humor, whole blood, and cerebrospinal and bronchoalveolar fluids) and significantly improved the performance of the diagnosis of toxoplasmosis. Taking into consideration all clinical samples, the mean gain in the crossing point value was 4.2 ± 1.7 cycles and was even more significant for amniotic fluid (5.8 ± 1.7 cycles).

Routine Identification of Medical Fungi by the New Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight System with a New Time-Effective Strategy

ABSTRACT We report here a clinical evaluation of the Vitek MS system for rapid fungal identification. A strategy that uses a single deposit without prior protein extraction was utilized to save time and money. Clinical isolates from the Toulouse University hospital were used to evaluate the performance of the Vitek MS compared to that of both routine laboratory techniques and Vitek2. The Vitek MS performed well in the identification of yeasts and Aspergillus fungi (93.2% of correct identifications).

Neonatal screening for congenital toxoplasmosis in a cohort of 165 women infected during pregnancy and influence of in utero treatment on the results of neonatal tests

The aim of this study was to determine the performances of methods used for the neonatal diagnosis of congenital toxoplasmosis. We included 165 pregnant women infected during pregnancy over a 10-year period. Fifty-seven cases of congenital toxoplasmosis were demonstrated (34.5%). Neonatal diagnosis gave positive results in 50 cases (88%). Parasites were isolated from placenta or cord blood in 61% of the infected newborns, more frequently from placenta (60%) than from cord blood (43%). This method was the only criterion of infection in 18% of these infected infants. The detection of specific IgM and IgA antibodies performed on 42 sera of infected infants allowed the diagnosis of congenital infection in 34 cases (81%). IgA antibodies were more frequently detected (60%) than specific IgM (50%). Neonatal and prenatal screening were carried out for 143 pregnant women. This combination diagnosed 39 of 40 infected infants (98%). Prenatal diagnosis identified 30 of 40 cases (75%). Nine cases were diagnosed through neonatal screening and one case with the postnatal follow-up. When prenatal diagnosis was positive, pyrimethamine and sulfadoxine were administered to the mothers (25 cases) in addition to spiramycin. Toxoplasma gondii was less frequently isolated in the placenta and the cord blood of these women (32% and 19%, respectively) than in women treated by spiramycin alone (83% and 63%) proving the antiparasitic action of these drugs. In conclusion, neonatal screening combining parasite detection in placenta and immunological methods on cord blood is essential particularly when prenatal diagnosis is negative. Therefore, when this diagnosis is positive, a treatment with pyrimethamine and sulfamide can be started in the first month of life.

Schistosomiasis Haematobium, Corsica, France

To the Editor: In Europe, urinary schistosomiasis (1) has previously been detected only in Portugal, where this focus disappeared during the 1950s (2). However, freshwater snails of the species Bulinus contortus, B. truncatus, and Planorbarius metidjensis, which are recognized intermediate hosts for Schistosoma haematobium trematodes, have been found in Portugal (3), Spain (4), and Corsica (5,6). This finding suggested that autochthonous schistosomiasis could re-emerge in southern Europe if these mollusks become infected. We report a probable focus for transmission of schistosomiasis haematobium in Corsica, France. In March 2014, a 4-year-old girl (index case-patient) from France was referred to the Toulouse University Hospital (Toulouse, France), with gross hematuria. Ultrasonography and cystoscopic examination of the bladder detected a polyp. Examination of the polyp for parasites identified bodies that were consistent with schistosome eggs. Parasitologic examination of urine confirmed schistosomiasis by detecting viable S. haematobium eggs. The parents of the girl (family A) did not report any stay or travel in an area to which urinary schistosomiasis was endemic; they reported summer holidays only in Mallorca in the Balearic Islands (Spain) and Corsica. However, her father reported that since 2012, he had experienced gross hematuria that had been evaluated by standard urologic investigations but not by cystoscopy; no etiology was determined. Parasitologic urinalysis in our hospital department showed numerous viable S. haematobium eggs in the father’s urine. The parents of the index case-patient also reported that an 8-year-old boy in a friend’s family (family B), who shared summer vacations with them had exhibited gross hematuria since February 2013. A third family (family C) was also investigated because they also spent holidays in Corsica with families A and B. Families B and C had also spent a summer in Mallorca, but they denied any contact with freshwater. Of 11 French native-born members of the 3 families, 6 had positive results for S. haematobium by urine examination. All case-patients had specific positive immunodiagnostic results by an ELISA that used S. mansoni extracts and by indirect hemagglutination. In addition, 2 family members who had a negative result by urine examination had a positive serologic result. Spending summer vacations in the same village in Corsica (Sainte-Lucie de Porto-Vecchio), where members of the 3 families had bathed at least once per holiday period in the Cavu River, was the epidemiologically prominent feature that linked these persons. Families A and C were in Sainte-Lucie de Porto-Vecchio in August 2011, and families A, B, and C were in the same location in August 2013. During these investigations, we were contacted by the Department of Tropical Medicine, Dusseldorf University Hospital (Dusseldorf, Germany), because a 10-year-old boy and his father had been given diagnoses of schistosomiasis haematobium on the basis of positive urinalysis results for S. haematobium eggs. Two other members of this family (5 persons) had a positive immunodiagnostic result. Locations of previous vacations for this family outside Germany included Spain (not the Balearic Islands) and Corsica, where they bathed frequently in the Cavu River. These epidemiologic findings provide strong circumstantial evidence supporting the presence of a previously unrecognized focus of urinary schistosomiasis in Corsica. We performed molecular analysis of schistosomal miracidia DNA. The second internal transcribed spacer region of the ribosomal gene complex (7,8) was amplified and sequenced. Viable eggs obtained from the patients in France were those of S. haematobium. Additional molecular investigations are being conducted to assess genetic diversity of this isolate from Corsica and the geographic origin of the introduced parasite. The malacologic situation in Sainte Lucie de Porto-Vecchio was investigated during May 12–19, 2014; three rivers (Figure) were included in the survey. Four sites were sampled in the Cavu River, and B. truncatus snails were found in 3 sites that corresponded to bathing areas (site 1: 41°43′53.57″N, 9°17′36.70″E; site 2: 41°43′22.13″N, 9°17′59.87″E; site 3: 41°42′8.40″N, 9°21′5.82″E). Snails were also found in the nearby Tarcu River (site 5) and Osu River (site 6). These findings confirmed previous data for the presence of B. truncatus snails in Corsica (5,6). Water temperature was recorded at 11:00 am at these 3 sites (range 15°C–16°C). This temperature range is not optimal for the snail intermediate host stage of the parasite life cycle (9,10). Of 148 live snails that were obtained in the Cavu River, none were infected with schistosome cercariae. Figure Corsica, France, showing malacologic survey sampling sites (oval) in 3 rivers (Tarcu, Cavu, and Osu). Bulinus truncatus snails were found at sites 1, 2, 3, 5, and 6. Data from the field survey and epidemiologic information for the cases in France and Germany, indicated transmission of schistosomiasis haematobium in the Cavu River in southeastern Corsica in 2011 and 2013. Additional supportive evidence is the fact that the father of the index case-patient had gross hematuria in 2012 and 2013. Two hypotheses are proposed to account for this situation. The first hypothesis is that the parasite (i.e., schistosome eggs) was transmitted by an infected person into the Cavu River in June or July 2011, when environmental conditions were favorable for snail infection. However, questions arise about survival of infected snails during the winter and their ability to reinfect the area during the following summers in 2012 and 2013. The second hypothesis is that schistosome eggs were spread by infected persons at the beginning of summer and caused a permanent transmission cycle in this focus. This situation would be difficult to control. Additional information should be obtained by a long-term malacologic survey to detect infected mollusks in this region.

Long-term outcome of children with congenital toxoplasmosis

OBJECTIVE Maternal toxoplasmosis infection acquired during pregnancy carries significant risk of fetal damage. We aimed to assess the long-term outcome of children and young adults with congenital toxoplasmosis diagnosed and treated in utero. STUDY DESIGN This was a 20 year prospective study (1985-2005). All mothers received spiramycin, alone or associated with pyrimethamine-sulfadoxine, and underwent amniocentesis and monthly ultrasound screening. Infected children were followed every 3-6 months. RESULTS Of 666 liveborn children (676 mothers), 112 (17%) had congenital toxoplasmosis. Among these, 107 were followed up for 12-250 months: 79 were asymptomatic (74%) and 28 had chorioretinitis (26%). Only 1 child had a serious neurological involvement. CONCLUSION The percentage of chorioretinitis in treated children depends on length of follow-up, but this complication occurs mainly before the age of 5 years and almost always before the age of 10 years. Visual impairment was infrequently severe, and outcome appears consistently good. Long-term follow-up is recommended to monitor ocular and neurological prognosis, whatever the practical difficulties.

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.

Imported Plasmodium knowlesi Malaria in a French Tourist Returning from Thailand

We report a case of imported Plasmodium knowlesi malaria in a French tourist following a vacation in Thailand. This case shows, first, tourists may contract knowlesi malaria even only staying on the beach and second, the diagnosis remains difficult, even with polymerase chain reaction methods.

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.

Prophylaxis of invasive aspergillosis with voriconazole or caspofungin during building work in patients with acute leukemia

Background Invasive aspergillosis is a common life-threatening infection in patients with acute leukemia. The presence of building work near to hospital wards in which these patients are cared for is an important risk factor for the development of invasive aspergillosis. This study assessed the impact of voriconazole or caspofungin prophylaxis in patients undergoing induction chemotherapy for acute leukemia in a hematology unit exposed to building work. Design and Methods This retrospective cohort study was carried out between June 2003 and January 2006 during which building work exposed patients to a persistently increased risk of invasive aspergillosis. This study compared the cumulative incidence of invasive aspergillosis in patients who did or did not receive primary antifungal prophylaxis. The diagnosis of invasive aspergillosis was based on the European Organization for Research and Treatment of Cancer/Mycosis Study Group criteria. Results Two-hundred and fifty-seven patients (213 with acute myeloid leukemia, 44 with acute lymphocytic leukemia) were included. The mean age of the patients was 54 years and the mean duration of their neutropenia was 21 days. Eighty-eight received antifungal prophylaxis, most with voriconazole (n=74). The characteristics of the patients who did or did not receive prophylaxis were similar except that pulmonary antecedents (chronic bronchopulmonary disorders or active tobacco use) were more frequent in the prophylaxis group. Invasive aspergillosis was diagnosed in 21 patients (12%) in the non-prophylaxis group and four (4.5%) in the prophylaxis group (P=0.04). Pulmonary antecedents, neutropenia at diagnosis and acute myeloid leukemia with high-risk cytogenetics were positively correlated with invasive aspergillosis, whereas primary prophylaxis was negatively correlated. Survival was similar in both groups. No case of zygomycosis was observed. The 3-month mortality rate was 28% in patients with invasive aspergillosis. Conclusions This study suggests that antifungal prophylaxis with voriconazole could be useful in acute leukemia patients undergoing first remission-induction chemotherapy in settings in which there is a high-risk of invasive aspergillosis.

Use of a Locked-Nucleic-Acid Oligomer in the Clamped-Probe Assay for Detection of a Minority Pfcrt K76T Mutant Population of Plasmodium falciparum

ABSTRACT Given the emergence of drug resistance and the high rate of polyclonal microorganism infections, the availability of a fast and sensitive test to detect minority mutant populations would be an improvement in the diagnosis of infectious diseases. A clamped-probe real-time PCR assay to diagnose the Plasmodium falciparum K76T mutation in clone populations was developed, using a wild-type-specific locked-nucleic-acid-containing oligomer to suppress wild-type PCR amplification and to enhance melting analysis with a mutation-specific detection probe.