Fungal infections in mechanically ventilated patients with COVID-19 during the first wave: the French multicentre MYCOVID study.

BACKGROUND: Patients with severe COVID-19 have emerged as a population at high risk of invasive fungal infections (IFIs). However, to our knowledge, the prevalence of IFIs has not yet been assessed in large populations of mechanically ventilated patients. We aimed to identify the prevalence, risk factors, and mortality associated with IFIs in mechanically ventilated patients with COVID-19 under intensive care. METHODS: We performed a national, multicentre, observational cohort study in 18 French intensive care units (ICUs). We retrospectively and prospectively enrolled adult patients (aged ≥18 years) with RT-PCR-confirmed SARS-CoV-2 infection and requiring mechanical ventilation for acute respiratory distress syndrome, with all demographic and clinical and biological follow-up data anonymised and collected from electronic case report forms. Patients were systematically screened for respiratory fungal microorganisms once or twice a week during the period of mechanical ventilation up to ICU discharge. The primary outcome was the prevalence of IFIs in all eligible participants with a minimum of three microbiological samples screened during ICU admission, with proven or probable (pr/pb) COVID-19-associated pulmonary aspergillosis (CAPA) classified according to the recent ECMM/ISHAM definitions. Secondary outcomes were risk factors of pr/pb CAPA, ICU mortality between the pr/pb CAPA and non-pr/pb CAPA groups, and associations of pr/pb CAPA and related variables with ICU mortality, identified by regression models. The MYCOVID study is registered with ClinicalTrials.gov, NCT04368221. FINDINGS: Between Feb 29 and July 9, 2020, we enrolled 565 mechanically ventilated patients with COVID-19. 509 patients with at least three screening samples were analysed (mean age 59·4 years [SD 12·5], 400 [79%] men). 128 (25%) patients had 138 episodes of pr/pb or possible IFIs. 76 (15%) patients fulfilled the criteria for pr/pb CAPA. According to multivariate analysis, age older than 62 years (odds ratio [OR] 2·34 [95% CI 1·39-3·92], p=0·0013), treatment with dexamethasone and anti-IL-6 (OR 2·71 [1·12-6·56], p=0·027), and long duration of mechanical ventilation (>14 days; OR 2·16 [1·14-4·09], p=0·019) were independently associated with pr/pb CAPA. 38 (7%) patients had one or more other pr/pb IFIs: 32 (6%) had candidaemia, six (1%) had invasive mucormycosis, and one (<1%) had invasive fusariosis. Multivariate analysis of associations with death, adjusted for candidaemia, for the 509 patients identified three significant factors: age older than 62 years (hazard ratio [HR] 1·71 [95% CI 1·26-2·32], p=0·0005), solid organ transplantation (HR 2·46 [1·53-3·95], p=0·0002), and pr/pb CAPA (HR 1·45 [95% CI 1·03-2·03], p=0·033). At time of ICU discharge, survival curves showed that overall ICU mortality was significantly higher in patients with pr/pb CAPA than in those without, at 61·8% (95% CI 50·0-72·8) versus 32·1% (27·7-36·7; p<0·0001). INTERPRETATION: This study shows the high prevalence of invasive pulmonary aspergillosis and candidaemia and high mortality associated with pr/pb CAPA in mechanically ventilated patients with COVID-19. These findings highlight the need for active surveillance of fungal pathogens in patients with severe COVID-19. FUNDING: Pfizer.

Clinical value of serology for the diagnosis of strongyloidiasis in travelers and migrants: A 4-year retrospective study using the Bordier IVD® Strongyloides ratti ELISA assay.

Strongyloides stercoralis serology is a sensitive method for strongyloidiasis diagnosis, but it is prone to cross-reactions with other helminthiases. This four-year retrospective study aimed at estimating the performance of the Bordier IVD® Strongyloides ratti ELISA assay in a non-endemic country (France). The study included all patients tested for strongyloidiasis in our center between 2015 and 2019, by both serology and stool examination. Cases were defined using an algorithm considering serological results, microscopic examination of stools, and other biological, clinical or epidemiological data. The study included 805 stools from 341 patients (70% migrants, 20% travelers, 10% without travel to a highly endemic area). Thirty patients (8.8%) had positive serology, 9 had microscopically proven strongyloidiasis, and 11 and 10 were classified as probable and possible strongyloidiasis, respectively. Performances of microscopy and serology were compared, considering proven and probable strongyloidiasis as true infections. The sensitivity, specificity, positive predictive value and negative predictive value of serology were 100%, 97%, 67% and 100%, respectively, and those of microscopic examination of stools were 45% (p < 0.01), 100% (p < 0.01), 100% (p = 0.079) and 96% (p < 0.001), respectively. Eosinophilia did not help in discriminating true-positive from false-positive results. Overall, these results underline the high value of the S. stercoralis serologic assay, compared to stool examination. The systematic use of this technique for screening purposes in travelers or migrants, or before onset of immunosuppressive therapy, could help to improve patient management and epidemiological knowledge.

TITLE: Utilité clinique de la sérologie pour le diagnostic de la strongyloïdose chez les voyageurs et les migrants : une étude rétrospective de 4 ans utilisant le test ELISA Strongyloides ratti Bordier IVD®. ABSTRACT: La sérologie de Strongyloides stercoralis est une méthode sensible pour le diagnostic de la strongyloïdose, mais elle est sujette à des réactions croisées avec d'autres helminthes. Cette étude rétrospective sur 4 ans visait à estimer les performances du test ELISA Strongyloides ratti Bordier IVD® dans un pays non endémique (la France). L'étude a inclus tous les patients testés pour la strongyloïdose dans notre centre entre 2015 et 2019, à la fois par sérologie et examen des selles. La définition des cas a été faite à l'aide d'un algorithme tenant compte des résultats sérologiques, de l'examen microscopique des selles et d'autres données biologiques, cliniques ou épidémiologiques. L'étude a inclus 805 selles de 341 patients (70 % de migrants, 20 % de voyageurs, 10 % sans voyage dans une zone de forte endémie). Trente patients (8,8 %) avaient une sérologie positive, 9 avaient une strongyloïdose prouvée au microscope, et 11 et 10 ont été classés respectivement comme strongyloïdose probable et possible. Les performances de la microscopie et de la sérologie ont été comparées, en considérant les strongyloïdoses avérées et probables comme de véritables infections. La sensibilité, la spécificité, la valeur prédictive positive et la valeur prédictive négative de la sérologie étaient de 100 %, 97 %, 67 % et 100 %, respectivement, et celles de l'examen microscopique des selles étaient de 45 % (p < 0,01), 100 % (p < 0,01), 100 % (p = 0,079) et 96 % (p < 0,001), respectivement. L'éosinophilie n'a pas aidé à distinguer les vrais positifs des faux positifs. Dans l'ensemble, ces résultats soulignent la valeur élevée du test sérologique de S. stercoralis, par rapport à l'examen des selles. L'utilisation systématique de cette technique à des fins de dépistage chez les voyageurs ou les migrants, ou avant le début d'un traitement immunosuppresseur, pourrait contribuer à améliorer la prise en charge des patients et les connaissances épidémiologiques.

Influenza- and COVID-19-Associated Pulmonary Aspergillosis: Are the Pictures Different?

Invasive pulmonary aspergillosis (IPA) in intensive care unit patients is a major concern. Influenza-associated acute respiratory distress syndrome (ARDS) and severe COVID-19 patients are both at risk of developing invasive fungal diseases. We used the new international definitions of influenza-associated pulmonary aspergillosis (IAPA) and COVID-19-associated pulmonary aspergillosis (CAPA) to compare the demographic, clinical, biological, and radiological aspects of IAPA and CAPA in a monocentric retrospective study. A total of 120 patients were included, 71 with influenza and 49 with COVID-19-associated ARDS. Among them, 27 fulfilled the newly published criteria of IPA: 17/71 IAPA (23.9%) and 10/49 CAPA (20.4%). Kaplan-Meier curves showed significantly higher 90-day mortality for IPA patients overall (p = 0.032), whereas mortality did not differ between CAPA and IAPA patients. Radiological findings showed differences between IAPA and CAPA, with a higher proportion of features suggestive of IPA during IAPA. Lastly, a wide proportion of IPA patients had low plasma voriconazole concentrations with a higher delay to reach concentrations > 2 mg/L in CAPA vs. IAPA patients (p = 0.045). Severe COVID-19 and influenza patients appeared very similar in terms of prevalence of IPA and outcome. The dramatic consequences on the patients' prognosis emphasize the need for a better awareness in these particular populations.

Is the COVID-19 Pandemic a Good Time to Include Aspergillus Molecular Detection to Categorize Aspergillosis in ICU Patients? A Monocentric Experience.

(1) Background: The diagnosis of invasive aspergillosis (IA) in an intensive care unit (ICU)remains a challenge and the COVID-19 epidemic makes it even harder. Here, we evaluatedAspergillus PCR input to help classifying IA in SARS-CoV-2-infected patients. (2) Methods: 45COVID-19 patients were prospectively monitored twice weekly for Aspergillus markers and anti-Aspergillus serology. We evaluated the concordance between (Ι) Aspergillus PCR and culture inrespiratory samples, and (ΙΙ) blood PCR and serum galactomannan. Patients were classified asputative/proven/colonized using AspICU algorithm and two other methods. (3) Results: Theconcordance of techniques applied on respiratory and blood samples was moderate (kappa = 0.58and kappa = 0.63, respectively), with a higher sensitivity of PCR. According to AspICU, 9/45 patientswere classified as putative IA. When incorporating PCR results, 15 were putative IA because theymet all criteria, probably with a lack of specificity in the context of COVID-19. Using a modifiedAspICU algorithm, eight patients were classified as colonized and seven as putative IA. (4)Conclusion: An appreciation of the fungal burden using PCR and Aspergillus serology was addedto propose a modified AspICU algorithm. This proof of concept seemed relevant, as it was inagreement with the outcome of patients, but will need validation in larger cohorts.

In vitro and in vivo immunomodulatory properties of octyl-ß-D-galactofuranoside during Leishmania donovani infection.

BACKGROUND: The chemotherapeutic arsenal available to treat visceral leishmaniasis is currently limited, in view of many drawbacks such as high cost, toxicity or emerging resistance. New therapeutic strategies are particularly needed to improve the management and the outcome in immunosuppressed patients. The combination of an immunomodulatory drug to a conventional anti-Leishmania treatment is an emerging concept to reverse the immune bias from Th2 to Th1 response to boost healing and prevent relapses. METHODS: Here, immunostimulating and leishmanicidal properties of octyl-ß-D-galactofuranose (Galf) were assessed in human monocyte-derived macrophages (HM) and in a murine model, after challenge with Leishmania donovani promastigotes. We recorded parasite loads and expression of various cytokines and immune effectors in HM and mouse organs (liver, spleen, bone marrow), following treatment with free (Galf) and liposomal (L-Galf) formulations. RESULTS: Both treatments significantly reduced parasite proliferation in HM, as well as liver parasite burden in vivo (Galf, P < 0.05). Consistent with in vitro results, we showed that Galf- and L-Galf-treated mice displayed an enhanced Th1 immune response, particularly in the spleen where pro-inflammatory cytokines TNF-α, IL-1ß and IL-12 were significantly overexpressed compared to control group. The hepatic recruitment of myeloid cells was also favored by L-Galf treatment as evidenced by the five-fold increase of myeloperoxidase (MPO) induction, which was associated with a higher number of MPO-positive cells within granulomas. By contrast, the systemic level of various cytokines such as IL-1ß, IL-6, IL-17A or IL-27 was drastically reduced at the end of treatment. CONCLUSIONS: Overall, these results suggest that Galf could be tested as an adjuvant in combination with current anti-parasitic drugs, to restore an efficient immune response against infection in a model of immunosuppressed mice.

A case report of photodermatoses induced by the antifungal drug terbinafine.

Terbinafine is an antifungaldrug(inhibitor of ergosterol synthesis) known to induce skin reactions. A 58-year-old female was treated with terbinafine for onychomycosis. On the fifth day of treatment a skin rash emerged on her sun-exposed areas. Biological testing did not find any allergic signs. Other skin reactions are well known with terbinafine, but we depict here, a case of photosensitisation induced by treatment, generally unknown for this antifungal. This side effect can be prevented by medication reconciliation and pharmaceutical advice from the clinical pharmacist.

Dibothriocephalus nihonkaiensis: an emerging foodborne parasite in Brittany (France)?

BACKGROUND: Diphyllobothriosis is an intestinal cestodosis caused by tapeworms of the family Diphyllobothriidae. In France, endemic cases are limited to south-east and due to Dibothriocephalus latus. In this paper, we investigate a series of seven cases of diphyllobothriosis in the non-endemic French region of Brittany. All have been diagnosed between 2016 and 2018 at the University Hospital of Rennes. METHODS: Parasites were identified by their morphological features and by phylogenetic analysis of the cox1 gene. Phylogenetic tree was built using maximum likelihood criterion under the GTR+G+I model and 2000 bootstrap replicates. A form was sent to all patients to collect data concerning clinical signs and possible sources of infection. RESULTS: All cases were due to Dibothriocephalus nihonkaiensis, a species strictly distributed in the North Pacific. Epidemiological investigation showed that the parasite was probably acquired in France, after consumption of Japanese food containing raw salmon. All patients presented with at least abdominal pain and fatigue except for one patient who had no symptoms. CONCLUSIONS: To our knowledge, this case series is the most important cohort of allochthonous diphyllobothriosis described in Europe. This sudden emergence raises concern about foodborne infections, highlighting (i) risky food habits in absence of adequate sanitary control; and (ii) the breaking of the rule of geographical restriction due to globalization and worldwide trades.

Treatment of toxoplasmosis: Current options and future perspectives.

Toxoplasmosis is a worldwide parasitic disease infecting about one third of humans, with possible severe outcomes in neonates and immunocompromised patients. Despite continuous and successful efforts to improve diagnosis, therapeutic schemes have barely evolved since many years. This article aims at reviewing the main clinical trials and current treatment practices, and at addressing future perspectives in the light of ongoing researches.

Comparison of three commercial multiplex PCR assays for the diagnosis of intestinal protozoa.

Although microscopic examination of stool samples remains the reference method for the diagnosis of intestinal protozoal infections, these techniques are time-consuming and require operators who are experienced and well trained. Molecular biology seems to offer performances at least equivalent in terms of sensitivity and specificity for certain parasites. This study aimed to compare three multiplex PCR assays on 93 prospectively collected positive stools (prospective cohort) and a panel of 12 more Cryptosporidium-positive samples (Cryptosporidium panel). On the prospective cohort, the sensitivity was 89%, 64% and 41% for Giardia sp. detection for BD MaxTM, G-DiaParaTM and RIDA®GENE, respectively and 75%, 100% and 100% for C. parvum/hominis detection. The sensitivity of the RIDA®GENE assay for all Cryptosporidium species was 100%, and for D. fragilis 71%. All the techniques obtained the same results for E. histolytica detection, with one positive sample. All species in the Cryptosporidium panel were identified by the RIDA®GENE PCR. The BD MaxTM and G-DiaParaTM assays detected only C. parvum/hominis with the exception of one positive sample for C. meleagridis. No assay showed satisfactory results for all parasites simultaneously, and the DNA extraction seems to be the critical step. More studies are needed to standardize this procedure.

TITLE: Comparaison de trois kits commerciaux de PCR multiplex pour la mise en évidence de protozoaires intestinaux. ABSTRACT: Bien que l'examen microscopique des selles reste la méthode de référence pour le diagnostic des protozooses intestinales, ces techniques sont chronophages et demandent une grande expérience et des opérateurs entrainés. La biologie moléculaire semble offrir des performances au moins équivalentes en termes de sensibilité comme de spécificité pour certains parasites. Cette étude visait à comparer trois techniques de PCR multiplex sur une cohorte de 93 selles positives collectées prospectivement et un panel de 12 échantillons positifs à Cryptosporidium. Respectivement pour BD MaxTM, G-DiaParaTM et RIDA®GENE la sensibilité était de 89 %, 64 % et 41 % pour la détection de Giardia sp. et 75 %, 100 % et 100 % pour la détection de C. parvum/hominis. La sensibilité de la technique RIDA®GENE pour l'ensemble des espèces de Cryptosporidium était de 100 % et de 71 % pour D. fragilis. Toutes les techniques ont obtenu les mêmes résultats pour la détection d'E. histolytica (1 échantillon positif). Toutes les espèces de Cryptosporidium ont été détectées par la PCR RIDA®GENE. Les techniques BD MaxTM et G-DiaParaTM ont détecté seulement C. parvum/hominis en dehors d'un échantillon positif à C. meleagridis. Aucun essai n'a montré de résultats satisfaisants pour l'ensemble des parasites simultanément et l'extraction d'ADN semble être l'étape critique. Plus d'études sont nécessaires afin de standardiser cette procédure.

Improving the diagnosis of invasive aspergillosis by the detection of Aspergillus in broncho-alveolar lavage fluid: Comparison of non-culture-based assays.

OBJECTIVES: This study aimed to evaluate new tools to diagnose invasive aspergillosis (IA) directly from broncho-alveolar lavage (BAL) samples. METHODS: All consecutive patients with suspected IA who underwent bronchoscopy with BAL were prospectively included. Mycological culture and ELISA detection of galactomannan (GM) were performed on BAL. Two in-house and two marketed PCR assays were used on BAL DNA extracts to detect Aspergillus species. Susceptibility testing was performed after culture; marketed PCR assays detected mutations in the CYP51A gene associated to resistance. RESULTS: Within 3 years, 1555 BAL samples were processed, including 413 samples from 387 immunosuppressed patients. IA diagnosis was no-IA, possible, probable or proven IA in 326, 23, 37 and 1 patients, respectively. PCR assays sensitivity for Aspergillus detection ranged from 61% to 74%, below GM (87%), but contrasting with 47% for cultures. Combining PCR to EORTC/MSG criteria increased the sensitivity to 100%. Interestingly, tests performance in non-hematological patients ranged from 60% to 75%, and were higher than in hematological patients, and those with prior exposure to antifungals. All 16 isolates of Aspergillus fumigatus were susceptible; PCR did not detect any resistance marker in the 37 A. fumigatus PCR-positive samples. CONCLUSION: The molecular detection of Aspergillus directly in BAL samples greatly improved the diagnosis of IA, particularly in non-hematological patients.

Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis.

Molecular diagnosis of congenital toxoplasmosis or disseminated toxoplasmosis is based mainly on PCR. The repeated DNA element rep529 has become the main DNA target used in most PCR methods, whether laboratory developed or commercial. In this multicenter study, we evaluated the Toxoplasma ELITe MGB (Elitech) commercial kit by comparison with three reference quantitative PCR assays (RAs) used routinely in three proficient laboratories of the French National Reference Center for Toxoplasmosis network, using Toxoplasma calibrated suspensions diluted to obtain a range of concentrations from 0.1 to 10,000 parasites/ml. These suspensions were extracted with either the DNA extraction kit (EXTRAblood; Elitech) recommended by the manufacturer or the QIAamp DNA minikit (Qiagen). The Toxoplasma ELITe MGB assay was also evaluated on a panel of 128 clinical samples, including 56 amniotic fluid samples, 55 placenta samples, and various other samples, of which 95 originated from patients with proven toxoplasmosis. The ELITe MGB assay amplified low-concentration replicates (<10 parasites/ml) of calibrated suspensions less frequently than the RAs of 2/3 laboratories. Additionally, the combination of EXTRAblood and Toxoplasma ELITe MGB yielded poorer sensitivity than the combination of QIAamp DNA minikit and ELITe MGB for low parasite concentrations (P < 0.001 for 1 parasite/ml). On clinical samples, the sensitivity and the specificity of the commercial assay were 89% and 100%, respectively. The sensitivity ranged from 79% (placenta samples) to 100% (amniotic fluid samples). Overall, this study shows that the Toxoplasma ELITe MGB assay is suitable for the diagnosis of toxoplasmosis from non-cell-rich or non-hemoglobin-rich samples and that the EXTRAblood kit is not optimal.

Epidemiology of Trichophyton verrucosum infection in Rennes University Hospital, France: A 12-year retrospective study.

Trichophyton verrucosum is a zoophilic dermatophyte, known as a causative agent of inflammatory mycoses of the skin and the scalp in humans. In this study, we reviewed all cases of T. verrucosum infection diagnosed in our laboratory over a 12-year period, to determine epidemiological and clinical characteristics. Among 18,340 samples analyzed, 5,186 cultured positive with dermatophytes (2674 patients), of which 64 samples (41 patients) were positive for T. verrucosum. Our data show that there was a strong influence of age on the type of lesion, with children and adults presenting more frequently with tinea capitis and skin infections, respectively (P < .0001). Infection of children and adults resulted more frequently from indirect and direct exposure to cattle, respectively (P < .01). We observed a marked increase of cases over the last 4 years, with a correlation of the number of cases and the mean annual rainfall (P < .05), suggesting that increasing humidity favors cattle infection, and thereby, human infection. Whether this increase is the consequence of climate changes remains to be determined but should be considered.

Synthesis and biological properties of galactofuranosyl-containing fluorescent dyes.

Two fluorescent galactofuranosides were synthesized and their biological activities evaluated on non-infected and Leishmania infected macrophages. Both tagged scaffolds were able to penetrate macrophages. Compared to the activity of the parent octyl galactofuranoside used as a reference, the fluorescein-conjugate showed altered biological properties while the rhodamine 6G one synergistically acted with the lipid chain to significantly increase antiparasitic activity.

A misleading false-negative result of Pneumocystis real-time PCR assay due to a rare punctual mutation: A French multicenter study.

This article describes a previously unreported mutation at position 210 (C210T) of the mitochondrial large subunit ribosomal RNA (mtLSUrRNA) gene of Pneumocystis jirovecii, which led to a false-negative result of a real-time polymerase chain reaction (PCR) assay. Since the aforementioned real-time PCR assay is widely used in France, a French multicenter study was conducted to estimate the mutation frequency and its potential impact on the routine diagnosis of Pneumocystis pneumonia (PCP). Through analysis of data obtained from eight centers, the mutation frequency was estimated at 0.28%. This low frequency should not call into question the routine use of this PCR assay. Nonetheless, the occurrence of the false-negative PCR result provides arguments for maintaining microscopic techniques combined to PCR assays to achieve PCP diagnosis.

Contribution of molecular tools for the diagnosis and epidemiology of fungal chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) rank second at chronic inflammatory diseases in industrialized countries and are an important public health concern. Diagnosis relies on a set of arguments including clinical signs, imaging, histopathologic and mycological analyses of sinus specimens, collected during nasal endoscopy. The sensitivity of fungal cultures is reported to be poor, even when direct examination is positive, thus the epidemiology of fungal chronic sinusitis is ill-known. This study evaluated the sensitivity of molecular diagnosis in 70 consecutive samples (61 patients with CRS) analysed at the University Hospital of Rennes during a 3-year period. DNA detection was performed using a conventional PCR method targeting the ITS1/ITS2 sequence and the resulting amplification products were sequenced. Fungal CRS was proven in 42 patients (69%), of which only 20 (48%) had a positive culture. 37/42 (88%) patients were diagnosed with a fungus ball, 3 with allergic fungal CRS and 2 with undetermined fungal CRS. PCR was positive in all 42 cases and direct sequencing allowed to identify fungi in all cases but one, and detected multiple infection in 3. Aspergillus fumigatus was present in 69% of patients; Cladosporium cladosporoides in 9.5%, Scedosporium sp., A. nidulans and A. flavus in 7% each. In 2/19 patients with negative direct examination, sequencing analysis revealed the presence of Capnobotryella sp. and C. cladosporoides, in clinical settings compatible with fungal sinusitis. In conclusion, ITS1/ITS2 PCR had a twice better sensitivity than culture, and combined sequencing provides accurate epidemiological data on fungal CRS.

Molecular diagnosis of toxoplasmosis in immunocompromised patients.

PURPOSE OF REVIEW: Toxoplasmosis in immunocompromised patients is associated with a high mortality rate. Molecular techniques are important tools to diagnose acute disease in immunocompromised patients, but there are various methods with variable efficiency. Some of them have been validated for the diagnosis of congenital toxoplasmosis, but the impact of their use has not been evaluated in immunocompromised patients. RECENT FINDINGS: Toxoplasmosis is of increasing importance in non-HIV immunocompromised patients. In addition, the picture of disease shows greater severity in South America, both in immunocompetent study participants and in congenitally infected infants. These epidemiological differences could influence the sensitivity of diagnostic methods. This review analyzes recent data on molecular diagnosis and compares them with older ones, in light of progress gained in molecular techniques and of recent epidemiological findings. Most recent studies were conducted in South America and used PCR targeting the B1 gene. PCR on blood could allow diagnosing a significant proportion of patients with ocular toxoplasmosis in Brazil. SUMMARY: Quantitative PCR methods with specific probes should be used to improve sensitivity and warrant specificity. Performance of quantitative PCR targeting the repeated 529 bp sequence for the diagnosis of toxoplasmosis in immunocompromised patients needs evaluation in field studies in South America and in western countries.

Identification, biochemical characterization, and in-vivo expression of the intracellular invertase BfrA from the pathogenic parasite Leishmania major.

The parasitic life cycle of Leishmania includes an extracellular promastigote stage that occurs in the gut of the insect vector. During that period, the sucrose metabolism and more specifically the first glycosidase of this pathway are essential for growth and survival of the parasite. We investigated the expression of the invertase BfrA in the promastigote and amastigote stages of three parasite species representative of the three various clinical forms and of various geographical areas, namely Leishmania major, L. donovani and L. braziliensis. Thereafter, we cloned, overexpressed and biochemically characterized this invertase BfrA from L. major, heterologously expressed in both Escherichia coli and L. tarentolae. For all species, expression levels of BfrA mRNA were correlated to the time of the culture and the parasitic stage (promastigotes > amastigotes). BfrA exhibited no activity when expressed as a glycoprotein in L. tarentolae but proved to be an invertase when not glycosylated, yet owing low sequence homology with other invertases from the same family. Our data suggest that BfrA is an original invertase that is located inside the parasite. It is expressed in both parasitic stages, though to a higher extent in promastigotes. This work provides new insight into the parasite sucrose metabolism.