Novel paradigm enables accurate monthly gestational screening to prevent congenital toxoplasmosis and more.

Background: Congenital toxoplasmosis is a treatable, preventable disease, but untreated causes death, prematurity, loss of sight, cognition and motor function, and substantial costs worldwide. Methods/Findings: In our ongoing USA feasibility/efficacy clinical trial, data collated with other ongoing and earlier published results proved high performance of an Immunochromatographic-test(ICT) that enables accurate, rapid diagnosis/treatment, establishing new paradigms for care. Overall results from patient blood and/or serum samples tested with ICT compared with gold-standard-predicate-test results found ICT performance for 4606 sera/1876 blood, 99.3%/97.5% sensitive and 98.9%/99.7% specific. However, in the clinical trial the FDA-cleared-predicate test initially caused practical, costly problems due to false-positive-IgM results. For 58 persons, 3/43 seronegative and 2/15 chronically infected persons had false positive IgM predicate tests. This caused substantial anxiety, concerns, and required costly, delayed confirmation in reference centers. Absence of false positive ICT results contributes to solutions: Lyon and Paris France and USA Reference laboratories frequently receive sera with erroneously positive local laboratory IgM results impeding patient care. Therefore, thirty-two such sera referred to Lyon's Reference laboratory were ICT-tested. We collated these with other earlier/ongoing results: 132 of 137 USA or French persons had false positive local laboratory IgM results identified correctly as negative by ICT. Five false positive ICT results in Tunisia and Marseille, France, emphasize need to confirm positive ICT results with Sabin-Feldman-Dye-test or western blot. Separate studies demonstrated high performance in detecting acute infections, meeting FDA, CLIA, WHO ASSURED, CEMark criteria and patient and physician satisfaction with monthly-gestational-ICT-screening. Conclusions/Significance: This novel paradigm using ICT identifies likely false positives or raises suspicion that a result is truly positive, rapidly needing prompt follow up and treatment. Thus, ICT enables well-accepted gestational screening programs that facilitate rapid treatment saving lives, sight, cognition and motor function. This reduces anxiety, delays, work, and cost at point-of-care and clinical laboratories. Author's Summary: Toxoplasmosis is a major health burden for developed and developing countries, causing damage to eyes and brain, loss of life and substantial societal costs. Prompt diagnosis in gestational screening programs enables treatment, thereby relieving suffering, and leading to > 14-fold cost savings for care. Herein, we demonstrate that using an ICT that meets WHO ASSURED-criteria identifying persons with/without antibody to Toxoplasma gondii in sera and whole blood with high sensitivity and specificity, is feasible to use in USA clinical practice. We find this new approach can help to obviate the problem of detection of false positive anti- T.gondii IgM results for those without IgG antibodies to T.gondii when this occurs in present, standard of care, predicate USA FDA cleared available assays. Thus, this accurate test facilitates gestational screening programs and a global initiative to diagnose and thereby prevent and treat T.gondii infection. This minimizes likelihood of false positives (IgG and/or IgM) while maintaining maximum sensitivity. When isolated IgM antibodies are detected, it is necessary to confirm and when indicated continue follow up testing in ∼2 weeks to establish seroconversion. Presence of a positive ICT makes it likely that IgM is truly positive and a negative ICT makes it likely that IgM will be a false positive without infection. These results create a new, enthusiastically-accepted, precise paradigm for rapid diagnosis and validation of results with a second-line test. This helps eliminate alarm and anxiety about false-positive results, while expediting needed treatment for true positive results and providing back up distinguishing false positive tests.

Multicentric Analysis of the Species Distribution and Antifungal Susceptibility of Clinical Isolates from Aspergillus Section Circumdati.

The clinical involvement and antifungal susceptibility of Aspergillus section Circumdati are poorly known. We analyzed 52 isolates, including 48 clinical isolates, belonging to 9 species inside the section Circumdati. The whole section exhibited, by the EUCAST reference method, a poor susceptibility to amphotericin B, but species/series-specific patterns were observed for azole drugs. This underlines the interest in getting an accurate identification inside the section Circumdati to guide the choice of antifungal treatment in clinical practice.

[Toxoplasmosis in pregnancy: Practical Management]. / Toxoplasmose pendant la grossesse : proposition actuelle de prise en charge pratique.

The burden of congenital toxoplasmosis has become small in France today, in particular as a result of timely therapy for pregnant women, fetuses and newborns. Thus, the French screening and prevention program has been evaluated and recently confirmed despite a decline over time in the incidence of toxoplasmosis. Serological diagnosis of maternal seroconversion is usually simple but can be difficult when the first trimester test shows the presence of IgM, requiring referral to an expert laboratory. Woman with confirmed seroconversion should be referred quickly to an expert center, which will decide with her on treatment and antenatal diagnosis. Although the level of proof is moderate, there is a body of evidence in favor of active prophylactic prenatal treatment started as early as possible (ideally within 3 weeks of seroconversion) to reduce the risk of maternal-fetal transmission, as well as symptoms in children. The recommended therapies to prevent maternal-fetal transmission are: (1) spiramycin in case of maternal infection before 14 gestational weeks; (2) pyrimethamine and sulfadiazine (P-S) with folinic acid in case of maternal infection at 14 WG or more. Amniocentesis is recommended to guide prenatal and neonatal care. If fetal infection is diagnosed by PCR on amniotic fluid, therapy with P-S should be initiated as early as possible or continued in order reduce the risk of damage to the brain or eyes. Further research is required to validate new approaches to preventing congenital toxoplasmosis.

Unexpected mould diversity in clinical isolates from French Guiana and associated identification difficulties.

New mold species are increasingly reported in invasive fungal infections. However, these fungi are often misdiagnosed or undiagnosed due to the use of inappropriate laboratory diagnostic tools. Tropical countries, such as French Guiana, harbor a vast diversity of environmental fungi representing a potential source of emerging pathogens. To assess the impact of this diversity on the accuracy of mold-infection diagnoses, we identified mold clinical isolates in French Guiana during a five-month follow-up using both microscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. In total, 38.8% of the 98 obtained molds isolates could not be identified and required a DNA-based identification. Fungal diversity was high, including 46 species, 26 genera, and 13 orders. Fungal ecology was unusual, as Aspergillus species accounted for only 27% of all isolates, and the Nigri section was the most abundant out of the six detected Aspergillus sections. Macromycetes (orders Agaricales, Polyporales, and Russulales) and endophytic fungi accounted for respectively 11% and 14% of all isolates. Thus, in tropical areas with high fungal diversity, such as French Guiana, routine mold identification tools are inadequate. Molecular identifications, as well as morphological descriptions, are necessary for the construction of region-specific mass spectrum databases. These advances will improve the diagnosis and clinical management of new fungal infections. LAY SUMMARY: In French Guiana, environmental fungal diversity may be a source of emerging pathogens. We evaluated microscopy and mass spectrometry to identify mold clinical isolates. With 39% of unidentified isolates, a region-specific mass spectrum database would improve the diagnosis of new fungal infections.

Multicentric Analysis of the Species Distribution and Antifungal Susceptibility of Cryptic Isolates from Aspergillus Section Fumigati.

The antifungal susceptibility of Aspergillus cryptic species is poorly known. We assessed 51 isolates, belonging to seven Fumigati cryptic species, by the EUCAST reference method and the concentration gradient strip (CGS) method. Species-specific patterns were observed, with high MICs for azole drugs, except for Aspergillus hiratsukae and Aspergillus tsurutae, and high MICs for amphotericin B for Aspergillus lentulus and Aspergillus udagawae Essential and categorical agreements between EUCAST and CGS results were between 53.3 and 93.3%.

Species Distribution and Comparison between EUCAST and Gradient Concentration Strips Methods for Antifungal Susceptibility Testing of 112 Aspergillus Section Nigri Isolates.

Aspergillus niger, the third species responsible for invasive aspergillosis, has been considered as a homogeneous species until DNA-based identification uncovered many cryptic species. These species have been recently reclassified into the Aspergillus section Nigri However, little is yet known among the section Nigri about the species distribution and the antifungal susceptibility pattern of each cryptic species. A total of 112 clinical isolates collected from 5 teaching hospitals in France and phenotypically identified as A. niger were analyzed. Identification to the species level was carried out by nucleotide sequence analysis. The MICs of itraconazole, voriconazole, posaconazole, isavuconazole, and amphotericin B were determined by both the EUCAST and gradient concentration strip methods. Aspergillus tubingensis (n = 51, 45.5%) and Aspergillus welwitschiae (n = 50, 44.6%) were the most common species while A. niger accounted for only 6.3% (n = 7). The MICs of azole drugs were higher for A. tubingensis than for A. welwitschiae The MIC of amphotericin B was 2 mg/liter or less for all isolates. Importantly, MICs determined by EUCAST showed no correlation with those determined by the gradient concentration strip method, with the latter being lower than the former (Spearman's rank correlation tests ranging from 0.01 to 0.25 depending on the antifungal agent; P > 0.4). In conclusion, A. niger should be considered as a minority species in the section Nigri The differences in MICs between species for different azoles underline the importance of accurate identification. Significant divergences in the determination of MIC between EUCAST and the gradient concentration strip methods require further investigation.

Comparison of matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) systems for the identification of moulds in the routine microbiology laboratory.

OBJECTIVES: The purpose of this study was to compare the efficiency of mould identification of two matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) systems - Vitek MS (VMS) and Microflex LT (MLT) - and the MSI application. METHODS: Moulds were collected retrospectively and prospectively to display epidemiological diversity of a microbiology laboratory. All of them were identified via sequencing. Strains were then identified using the VMS v3.0, the MLT, and the MSI software applied on MLT spectra. Rates of correct identifications to the species, to the complex, and to the genus level were compared with the molecular reference standard. RESULTS: A total of 102 isolates were collected. The rate of correct identification to the species level with the MLT was 42.2% (43/102) with a threshold of 1.7 (vs. 16.7% (17/102) with a threshold of 2.0, p < 0.05). The VMS performed better than the MLT with a threshold of 1.7 for species (49.0% (50/102), p 0.33) and complex level identifications (71.6% (73/102) vs. 54.9% (56/102), p < 0.05). However the highest performances were observed when the MLT spectra were analysed via the Mass Spectrometry Identification (MSI) software reaching 90.2% (92/102) of correct identification to the species, 92.2% (94/102) to the species complex and 94.1% (96/102) to the genus level. CONCLUSIONS: The VMS performed better than the MLT for mould identification. However, it remains of utmost importance to expand commercial databases, as performances of the MLT highly improved when using the MSI software and its extended database, reaching far above the VMS system. Thus the VMS could benefit from the use of this online tool.

Nucleotide Sequence Database Comparison for Routine Dermatophyte Identification by Internal Transcribed Spacer 2 Genetic Region DNA Barcoding.

Conventional dermatophyte identification is based on morphological features. However, recent studies have proposed to use the nucleotide sequences of the rRNA internal transcribed spacer (ITS) region as an identification barcode of all fungi, including dermatophytes. Several nucleotide databases are available to compare sequences and thus identify isolates; however, these databases often contain mislabeled sequences that impair sequence-based identification. We evaluated five of these databases on a clinical isolate panel. We selected 292 clinical dermatophyte strains that were prospectively subjected to an ITS2 nucleotide sequence analysis. Sequences were analyzed against the databases, and the results were compared to clusters obtained via DNA alignment of sequence segments. The DNA tree served as the identification standard throughout the study. According to the ITS2 sequence identification, the majority of strains (255/292) belonged to the genus Trichophyton, mainly T. rubrum complex (n = 184), T. interdigitale (n = 40), T. tonsurans (n = 26), and T. benhamiae (n = 5). Other genera included Microsporum (e.g., M. canis [n = 21], M. audouinii [n = 10], Nannizzia gypsea [n = 3], and Epidermophyton [n = 3]). Species-level identification of T. rubrum complex isolates was an issue. Overall, ITS DNA sequencing is a reliable tool to identify dermatophyte species given that a comprehensive and correctly labeled database is consulted. Since many inaccurate identification results exist in the DNA databases used for this study, reference databases must be verified frequently and amended in line with the current revisions of fungal taxonomy. Before describing a new species or adding a new DNA reference to the available databases, its position in the phylogenetic tree must be verified.

Species Identification and In Vitro Antifungal Susceptibility of Aspergillus terreus Species Complex Clinical Isolates from a French Multicenter Study.

Aspergillus section Terrei is a species complex currently comprised of 14 cryptic species whose prevalence in clinical samples as well as antifungal susceptibility are poorly known. The aims of this study were to investigate A. Terrei clinical isolates at the species level and to perform antifungal susceptibility analyses by reference and commercial methods. Eighty-two clinical A. Terrei isolates were collected from 8 French university hospitals. Molecular identification was performed by sequencing parts of beta-tubulin and calmodulin genes. MICs or minimum effective concentrations (MECs) were determined for 8 antifungal drugs using both EUCAST broth microdilution (BMD) methods and concentration gradient strips (CGS). Among the 79 A. Terrei isolates, A. terreus stricto sensu (n = 61), A. citrinoterreus (n = 13), A. hortai (n = 3), and A. alabamensis (n = 2) were identified. All strains had MICs of ≥1 mg/liter for amphotericin B, except for two isolates (both A. hortai) that had MICs of 0.25 mg/liter. Four A. terreus isolates were resistant to at least one azole drug, including one with pan-azole resistance, yet no mutation in the CYP51A gene was found. All strains had low MECs for the three echinocandins. The essential agreements (EAs) between BMD and CGS were >90%, except for those of amphotericin B (79.7%) and itraconazole (73.4%). Isolates belonging to the A section Terrei identified in clinical samples show wider species diversity beyond the known A. terreus sensu stricto Azole resistance inside the section Terrei is uncommon and is not related to CYP51A mutations here. Finally, CGS is an interesting alternative for routine antifungal susceptibility testing.

Validation of a New Web Application for Identification of Fungi by Use of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry has emerged as a reliable technique to identify molds involved in human diseases, including dermatophytes, provided that exhaustive reference databases are available. This study assessed an online identification application based on original algorithms and an extensive in-house reference database comprising 11,851 spectra (938 fungal species and 246 fungal genera). Validation criteria were established using an initial panel of 422 molds, including dermatophytes, previously identified via DNA sequencing (126 species). The application was further assessed using a separate panel of 501 cultured clinical isolates (88 mold taxa including dermatophytes) derived from five hospital laboratories. A total of 438 (87.35%) isolates were correctly identified at the species level, while 26 (5.22%) were assigned to the correct genus but the wrong species and 37 (7.43%) were not identified, since the defined threshold of 20 was not reached. The use of the Bruker Daltonics database included in the MALDI Biotyper software resulted in a much higher rate of unidentified isolates (39.76 and 74.30% using the score thresholds 1.7 and 2.0, respectively). Moreover, the identification delay of the online application remained compatible with real-time online queries (0.15 s per spectrum), and the application was faster than identifications using the MALDI Biotyper software. This is the first study to assess an online identification system based on MALDI-TOF spectrum analysis. We have successfully applied this approach to identify molds, including dermatophytes, for which diversity is insufficiently represented in commercial databases. This free-access application is available to medical mycologists to improve fungal identification.

Interpretation of the Elecsys Toxo IgG avidity results for very low and very high index: study on 741 sera with a determined date of toxoplasmosis.

Initial results with the Elecsys Toxo IgG Avidity assay showed some potential for interpretation of a very low or very high index result. We aimed to examine these new insights into interpretation using a large panel of serum samples and to define the optimal thresholds. A total of 741 patient serum samples with known date of infection (from a few weeks to more than 9 months after infection), were analysed with the Elecsys Toxo IgG Avidity assay. Values ≥80% (threshold defined by the manufacturer) were reported in 289 sera; 288 sera were sampled more than 4 months after infection. Thus, avidity values ≥80% excluded an infection less than 4 months. Avidity values ≥90% were reported in 112 sera sampled more than 9 months after infection. Thus, avidity values ≥90% excluded infection less than 9 months. Moreover avidity values ≤15% were reported in the 62 sera sampled less than 3 months after infection. Thus avidity values ≤15% excluded infection more than 3 months.

[Targeting abattoirs to control cystic echinococcosis in Algeria]. / Le ciblage des abattoirs dans la lutte contre l'échinococcose kystique en Algérie.

Cystic echinococcosis (CE) is an important anthropozoonotic parasitic common in Algeria. The predominant life cycle of E. granulosus is a synanthropic cycle with domestic dogs as definitive hosts and livestock animals as intermediate hosts. Slaughter activity represents a potential source for dogs to access infected offal. The aim of the present study was to determine if the contact between dogs and potentially infected offal was possible in licensed abattoirs. Eighty-one private and public abattoirs located in eastern Algeria were assessed with respect to their level of protection against the intrusion of dogs.We have demonstrated that in 42 % of these abattoirs, dogs could easily come in contact with potentially parasitized offal. The most common incorrect practices were the dumping of offal freely into the environment, the feeding of dogs with offal, and the leaving of unattended offal in an unsealed chamber. Overall, some hazardous practices remained common customs of workers, and enough abattoirs remain non-compliant that the cattledog domestic cycle of CE is unlikely to be broken. Hence, some recommended measures to interrupt parasite transmission include the following: recognition of the importance of abattoirs in the maintenance of canine echinococcosis, the controlled and proper disposal of offal, the abolishment of the custom of feeding dogs with infected offal and improvements in the level of health education of abattoir staff.

Landscape and climatic characteristics associated with human alveolar echinococcosis in France, 1982 to 2007.

Human alveolar echinococcosis (AE) is a severe hepatic disease caused by Echinococcus multilocularis. In France, the definitive and intermediate hosts of E. multilocularis (foxes and rodents, respectively) have a broader geographical distribution than that of human AE. In this two-part study, we describe the link between AE incidence in France between 1982 and 2007 and climatic and landscape characteristics. National-level analysis demonstrated a dramatic increase in AE risk in areas with very cold winters and high annual rainfall levels. Notably, 52% (207/401) of cases resided in French communes (smallest French administrative level) with a mountain climate. The mountain climate communes displayed a 133-fold (95% CI: 95-191) increase in AE risk compared with communes in which the majority of the population resides. A case-control study performed in the most affected areas confirmed the link between AE risk and climatic factors. This arm of the study also revealed that populations residing in forest or pasture areas were at high risk of developing AE. We therefore hypothesised that snow-covered ground may facilitate predators to track their prey, thus increasing E. multilocularis biomass in foxes. Such climatic and landscape conditions could lead to an increased risk of developing AE among humans residing in nearby areas.

MALDI-TOF typing highlights geographical and fluconazole resistance clusters in Candida glabrata.

Utilizing matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra for Candida glabrata typing would be a cost-effective and easy-to-use alternative to classical DNA-based typing methods. This study aimed to use MALDI-TOF for the typing of C. glabrata clinical isolates from various geographical origins and test its capacity to differentiate between fluconazole-sensitive and -resistant strains.Both microsatellite length polymorphism (MLP) and MALDI-TOF mass spectra of 58 C. glabrata isolates originating from Marseilles (France) and Tunis (Tunisia) as well as collection strains from diverse geographic origins were analyzed. The same analysis was conducted on a subset of C. glabrata isolates that were either susceptible (MIC ≤ 8 mg/l) or resistant (MIC ≥ 64 mg/l) to fluconazole.According to the seminal results, both MALDI-TOF and MLP classifications could highlight C. glabrata population structures associated with either geographical dispersal barriers (p < 10(-5)) or the selection of antifungal drug resistance traits (<10(-5)).In conclusion, MALDI-TOF geographical clustering was congruent with MPL genotyping and highlighted a significant population genetic structure according to fluconazole susceptibility in C. glabrata. Furthermore, although MALDI-TOF and MLP resulted in distinct classifications, MALDI-TOF also classified the isolates with respect to their fluconazole susceptibility profile. Further prospective studies are required to evaluate the capacity of MALDI-TOF typing to investigate C. glabrata infection outbreaks and predict the antifungal susceptibility profile of clinical laboratory isolates.

Comparison of MALDI-TOF mass spectra with microsatellite length polymorphisms in Candida albicans.

Candida albicans is the most frequent yeast involved in human infections. Its population structure can be divided into several genetic clades, some of which have been associated with antifungal susceptibility. Therefore, detecting and monitoring fungal clones in a routine laboratory setting would be a major epidemiological advance. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra results are now widely used as bar codes to identify microorganisms in clinical microbiology laboratories. This study aimed at testing MALDI-TOF mass spectra bar codes to identify clades among a set of C. albicans isolates. Accordingly, 102 clinical strains were genotyped using 10 microsatellite markers and analyzed via MALDI-TOF mass spectrometry. The mass spectra were compared with a reference spectral library including 33 well-characterized collection strains, using a Microflex(TM) system and Biotyper(TM) software, to test the capacity of the spectrum of a given isolate to match with the reference mass spectrum of an isolate from the same genetic clade. Despite high confidence species identification, the spectra failed to significantly match with the corresponding clade (p = 0.74). This was confirmed with the MALDI-TOF spectra similarity dendrogram, in which the strains were dispersed irrespective of their genetic clade. Various attempts to improve intra-clade spectra recognition were unsuccessful. In conclusion, MALDI-TOF mass spectra bar code analysis failed to reliably recognize genetically related C. albicans isolates. Further studies are warranted to develop alternative MALDI-TOF mass spectra analytical approaches to identify and monitor C. albicans clades in the routine clinical laboratory.

Evaluation of the Aspergillus Western blot IgG kit for diagnosis of chronic aspergillosis.

Immunoprecipitin detection (IPD) is the current reference confirmatory technique for anti-Aspergillus antibody detection; however, the lack of standardization is a critical drawback of this assay. In this study, we evaluated the performance of the Aspergillus Western blot (Asp-WB) IgG kit (LDBio Diagnostics, Lyon, France), a recently commercialized immunoblot assay for the diagnosis of various clinical presentations of chronic aspergillosis. Three hundred eight serum samples from 158 patients with aspergillosis sensu lato (s.l.) were analyzed. More specifically, 267 serum samples were derived from patients with Aspergillus disease, including 89 cases of chronic pulmonary aspergillosis, 10 of aspergilloma, and 32 of allergic bronchopulmonary aspergillosis, while 41 samples were from patients with Aspergillus colonization, including 15 cystic fibrosis (CF) and 12 non-CF patients. For blood donor controls, the Asp-WB specificity was 94%, while the kit displayed a sensitivity for the aspergillosis s.l. diagnosis of 88.6%, with a diagnostic odds ratio (DOR) of 119 (95% confidence interval [CI], 57 to 251). The DOR values were 185.22 (95% CI,78.79 to 435.45) and 43.74 (95% CI, 15.65 to 122.20) for the diagnosis of Aspergillus disease and Aspergillus colonization, respectively. Among the patients, the sensitivities of the Asp-WB in the diagnosis of Aspergillus colonization were 100% and 41.7% in CF and non-CF patients, respectively. The Asp-WB yielded fewer false-negative results than did IPD. In conclusion, the Asp-WB kit performed well for the diagnosis of various clinical presentations of aspergillosis in nonimmunocompromised patients, with an enhanced standardization and a higher sensitivity than with IPD, which is the current reference method.

High dermatophyte contamination levels in hairdressing salons of a West African suburban community.

Tinea capitis is a dermatophyte infection of scalp is commonly spread by currently infected patients, asymptomatic carriers or by fomites, such as hairdressing tools. However, studies on the risk factors of Tinea capitis remain scarce. The aim of this study was to evaluate the dermatophytes contamination level of the hairdressing tools to which hairdressing salon customers are exposed in Sirakoro-Méguétana, a suburb of Bamako, the capital city of Mali. A total of 41 hairdressing tools were sampled in five hairdressing salons. Two anthropophilic dermatophytes species, Microsporum audouinii (53.3%) and Trichophyton soudanense (46.7%), were cultured from 30 (73.2%) samples. This first study, addressing hairdressing salons dermatophyte contamination, revealed a strikingly high contamination of hairdressing tools with dermatophyte propagules, which exposes hairdressing salons customers to an important dermatophytosis risk. The sterilisation of hairdressing tools is central to preventing dermatophytoses spreading. Appropriate community information and hairdressers training should be implemented in this view.