Molecular Study of Toxoplasma gondii Isolates Originating from Humans and Organic Pigs in Belgium.

Toxoplasma gondii is a worldwide prevalent, zoonotic parasite of major importance for public health, which can infect any warm-blooded animal species, including humans. Humans can get infected by consumption of meat from a chronically infected animal, by ingestion of sporulated oocysts (resulting from the sexual replication in felids), via contaminated water, soil, or vegetables, and by vertical transmission via the placenta. Infection through meat consumption is estimated to be one of the main sources of human toxoplasmosis cases in developed countries, and more specifically pork is considered to be responsible for 41% of foodborne human toxoplasmosis cases in the United States. To better assess the role of pork as a source of T. gondii infection in humans in Belgium, parasites were isolated from pigs to compare with human clinical isolates in a molecular epidemiological study. A positive result was obtained by magnetic capture-quantitative polymerase chain reaction for T. gondii in 14 out of the 92 hearts sampled during 2016 and 2017 from pigs raised in organic farms. From 9 of these 14 samples, parasites were isolated by mouse bioassay, demonstrating the presence of viable T. gondii in animals intended for human consumption. When genotyped and compared with 15 human isolates obtained during 2015 and 2016, a highly related structured population was demonstrated. Overall, these findings demonstrate the presence of infectious T. gondii in pigs intended for human consumption. Therefore, a potential transmission of T. gondii strains from pigs to humans could occur. However, both species could also be infected via a common source of infection such as oocysts. Furthermore, Belgium does not have an official surveillance program for T. gondii in human cases or food-producing animals; as a consequence, the detection of the infection source of a patient is very rare. Overall, this study reinforces the identification of pork as a potential risk for the consumers.

Aspergillus pseudodeflectus: a new human pathogen in liver transplant patients.

BACKGROUND: Liver transplant recipients are at high risk of developing invasive aspergillosis and in particular by Aspergillus fumigatus which is the most commonly encountered species in this population. Other non-fumigatus Aspergillus species with reduced susceptibility to antifungal drugs can also be involved. Accurate identification associated to antifungal susceptibility testing is essential for therapy adjustment. We report a case of invasive pulmonary aspergillosis due to Aspergillus pseudodeflectus in a liver transplant recipient. To our knowledge, this is the first reported case of invasive aspergillosis due to this species with a reduced susceptibility to azoles. CASE PRESENTATION: A 64 year-old woman with drug-induced fulminant hepatitis underwent liver transplantation. Prophylactic treatment with caspofungin was introduced due to aspergillosis risk factors consisting in hemodialysis and fulminant hepatitis. Six weeks after transplantation, CT scan showed a right pulmonary opacity associated with an increase of galactomannan (index 5.4). Culture of BAL grew with several colonies of Aspergillus sp. The diagnosis of invasive aspergillosis was probable according to the EORTC criteria. The antifungal susceptibility tests (Etest®) revealed low MICs to echinocandins and amphotericin B) but high MICs to azoles. After these results, voriconazole was switched to liposomal amphotericin B. The patient died one month after diagnosis from a refractory septic shock with multiple organ failure. A molecular identification of isolate, based on partial ß-tubulin and calmodulin genes, was performed and identified A. pseudodeflectus. CONCLUSIONS: Our case raises the question of pathogenicity of this species, which belongs to Aspergillus section Usti and is genetically and morphologically very close to Aspergillus calidoustus that was previously reported in human transplant recipients.

Added Value of Next-Generation Sequencing for Multilocus Sequence Typing Analysis of a Pneumocystis jirovecii Pneumonia Outbreak1.

Pneumocystis jirovecii is a major threat for immunocompromised patients, and clusters of pneumocystis pneumonia (PCP) have been increasingly described in transplant units during the past decade. Exploring an outbreak transmission network requires complementary spatiotemporal and strain-typing approaches. We analyzed a PCP outbreak and demonstrated the added value of next-generation sequencing (NGS) for the multilocus sequence typing (MLST) study of P. jirovecii strains. Thirty-two PCP patients were included. Among the 12 solid organ transplant patients, 5 shared a major and unique genotype that was also found as a minor strain in a sixth patient. A transmission map analysis strengthened the suspicion of nosocomial acquisition of this strain for the 6 patients. NGS-MLST enables accurate determination of subpopulation, which allowed excluding other patients from the transmission network. NGS-MLST genotyping approach was essential to deciphering this outbreak. This innovative approach brings new insights for future epidemiologic studies on this uncultivable opportunistic fungus.

MALDI-TOF mass spectrometry identification of filamentous fungi in the clinical laboratory.

This study aimed to validate the effectiveness of a standardised procedure for the MALDI-TOF mass spectrometry (MS)-based identification on a large sample of filamentous fungi routinely identified in university hospitals' laboratories. Non-dermatophyte filamentous fungi prospectively isolated in the routine activity of five teaching hospitals in France were first identified by conventional methods in each laboratory and then by MS in one centre. DNA sequence-based identification resolved discrepancies between both methods. In this study, of the 625 analysed filamentous fungi of 58 species, 501 (80%) and 556 (89%) were correctly identified by conventional methods and MS respectively. Compared with the conventional method, MS dramatically enhanced the performance of the identification of the non-Aspergillus filamentous fungi with a 31-61% increase in correct identification rate. In conclusion, this study on a large sample of clinical filamentous fungi taxa demonstrates that species identification is significantly improved by MS compared with the conventional method. The main limitation is that MS identification is possible only if the species is included in the reference spectra library. Nevertheless, for the routine clinical laboratory, MS provides the means to attain markedly accurate results in filamentous fungi identification, which was previously restricted to only a few reference laboratories.

Minimum inhibitory concentrations of amphotericin B against Candida krusei isolates from a French teaching hospital laboratory: a retrospective study over 8 years.

Recent studies have shown decreased susceptibility of Candida krusei to amphotericin B (AmB), in addition to its inherent resistance to fluconazole. The susceptibility of C. krusei to AmB was studied in the Parasitology-Mycology laboratory of Grenoble Teaching Hospital, France. Between 2003 and 2011, we analysed 200 C. krusei isolates from 130 patients. The isolates were mainly collected in intensive care, cardio-thoracic and cancer/haematology units. Minimum inhibitory concentrations (MICs) were determined by the E-test method. The modal MIC was 0.5 µg ml(-1); the MIC(50) and MIC(90) (MICs encompassing 50% and 90% of all isolates tested, respectively) were 0.5 µg ml(-1) and 1 µg ml(-1). The Cuzick's and Kendall's tests showed a significant increase in MIC values between 2003 and 2011 (P = 0.001 and P ≤ 0.001, respectively), regardless of age or gender. No statistical difference was reached with these tests when the first 100 or 50 data were excluded. Despite the increase observed in the first period of the study, our results confirm the low AmB MICs reported in previous studies. However, some authors have recently reported much higher MICs. This discrepancy cannot be explained by method biases and could reflect C. krusei epidemiological differences among populations.

Factors influencing the microbial composition of metalworking fluids and potential implications for machine operator's lung.

Hypersensitivity pneumonitis, also known as "machine operator's lung" (MOL), has been related to microorganisms growing in metalworking fluids (MWFs), especially Mycobacterium immunogenum. We aimed to (i) describe the microbiological contamination of MWFs and (ii) look for chemical, physical, and environmental parameters associated with variations in microbiological profiles. We microbiologically analyzed 180 MWF samples from nonautomotive plants (e.g., screw-machining or metal-cutting plants) in the Franche-Comté region in eastern France and 165 samples from three French automotive plants in which cases of MOL had been proven. Our results revealed two types of microbial biomes: the first was from the nonautomotive industry, showed predominantly Gram-negative rods (GNR), and was associated with a low risk of MOL, and the second came from the automotive industry that was affected by cases of MOL and showed predominantly Gram-positive rods (GPR). Traces of M. immunogenum were sporadically detected in the first type, while it was highly prevalent in the automotive sector, with up to 38% of samples testing positive. The use of chromium, nickel, or iron was associated with growth of Gram-negative rods; conversely, growth of Gram-positive rods was associated with the absence of these metals. Synthetic MWFs were more frequently sterile than emulsions. Vegetable oil-based emulsions were associated with GNR, while mineral ones were associated with GPR. Our results suggest that metal types and the nature of MWF play a part in MWF contamination, and this work shall be followed by further in vitro simulation experiments on the kinetics of microbial populations, focusing on the phenomena of inhibition and synergy.

Biosurveillance of Selected Pathogens with Zoonotic Potential in a Zoo.

Monitoring of infectious diseases is one of the most important pillars of preventive medicine in zoos. Screening for parasitic and bacterial infections is important to keep animals and equipment safe from pathogens that may pose a risk to animal and human health. Zoos usually contain many different animal species living in proximity with people and wild animals. As an epidemiological probe, 188 animals (122 mammals, 65 birds, and one reptile) from a zoo in Slovenia were examined for selected pathogens. Antibodies to Toxoplasma gondii and Neospora caninum were detected by ELISA in 38% (46/122) and 3% (4/122) of mammals, and in 0% (0/64) and 2% (1/57) of birds, respectively; the reptile (0/1) was negative. A statistically significant difference in T. gondii prevalence was found in Carnivora compared to Cetartiodactyla and primate antibodies to Encephalitozoon cuniculi were detected by IFAT in 44% (52/118) of mammals and 20% (11/56) of birds, respectively; the reptile (0/1) was negative. Herbivores had a higher chance of being infected with E. cuniculi compared to omnivores. Antibodies to Chlamydia abortus and Coxiella burnetii were not detected in any of the 74 tested zoo animals. The sera of 39 wild rodents found in the zoo were also examined; they were negative for all three parasites. The parasite T. gondii was detected by PCR in the tissue of two mute swans (Cygnus olor), three eastern house mice (Mus musculus), one yellow-necked field mouse (Apodemus flavicollis), and one striped field mouse (A. agrarius). Positive samples were genotyped by a single multiplex PCR assay using 15 microsatellite markers; one sample from a mute swan was characterized as type II. This micro-epidemiological study offers a better understanding of pathogens in zoo animals and an understanding of the role of zoos in biosurveillance.

Molecular Evidence of Toxoplasma gondii, Neospora caninum, and Encephalitozoon cuniculi in Red Foxes ( Vulpes vulpes).

Toxoplasma gondii, Neospora caninum, and Encephalitozoon cuniculi are important infectious agents, with T. gondii and E. cuniculi having zoonotic potential. There are two main clonal lineages (types I and II) of T. gondii in Europe, but little is known about genotypes of T. gondii in wild animals. The aim of our study was molecular detection of these three pathogens in tissues of wild red foxes ( Vulpes vulpes) from the Czech Republic. Using PCR (B1 gene), we detected T. gondii in 10% of the animals that we tested ( n=100); N. caninum and E. cuniculi were not detected. The T. gondii samples were genotyped by single multiplex PCR assay with 15 microsatellite markers. Five samples were successfully genotyped as genotype II, a unique finding for T. gondii isolated from red foxes from the Czech Republic.

Molecular detection of Toxoplasma gondii and Neospora caninum in birds from South Africa.

There are not any records on the detection of Toxoplasma gondii and Neospora caninum in tissues of wild birds in the African continent. The aim of the study was to investigate the occurrence of DNA from these protozoan parasites in brain tissue samples collected in years 2014-2015 from 110 wild and domestic birds of 15 orders. Birds came mainly from the province of Limpopo (n=103); the other seven birds came from other five provinces of South Africa. Parasite DNAs were detected by PCR in animal brains. While all samples were negative for N. caninum, T. gondii DNA was detected in three (2.7%) birds: a Red-eyed Dove (Streptopelia semitorquata), a Laughing Dove (S. senegalensis) and a Southern-Yellow-billed Hornbill (Tockus leucomelas), all from Limpopo province. Positive samples were selected for genotyping by a 15 microsatellite markers method in a single multiplex PCR assay. Only the sample from the Red-eyed Dove was successfully genotyped and characterized as type II. This is the first detection of T. gondii in tissue of native African wild birds and the first study focusing on N. caninum in birds from South Africa.

Prevalence of toxoplasmosis and genetic characterization of Toxoplasma gondii strains isolated in wild birds of prey and their relation with previously isolated strains from Turkey.

Toxoplasma gondii is a protozoon parasite that causes congenital toxoplasmosis, as well as other serious clinical presentations, in immune compromised humans. Analyses of the prevalence and genotyping of strains from the definitive host and intermediate hosts will help to understanding the circulation of the different strains and elucidating the role of the genotype(s) in human toxoplasmosis. Turkey has a specific geographic location bridging Africa, Europe, and Asia. We hypothesized that T. gondii strains may have been transferred to Turkey from these continents via migratory birds or vice versa. The present study aimed to assess the prevalence of toxoplasmosis in wild birds of prey of Izmir and Manisa provinces as well as genetically characterize T. gondii strains from these wild birds to show the relation between bird strains and neighboring stray cats as well as human strains previously isolated in Turkey. Tissues obtained from 48 wild birds were investigated for the presence of T. gondii DNA and then bioassayed in mouse. Isolated strains were genotyped using 15 microsatellite markers. The prevalence of T. gondii DNA was found to be 89.6% (n: 43/48) in wild birds. Out of 43 positive samples, a total of 14 strains were genotyped by 15 microsatellite markers. Among them, eight were type II, three were type III and three were mixture of genotypes (two type II/II and one was II/III). These are the first data that showed the presence of T. gondii and types II and III genotypes in wild birds of Turkey. Moreover, Africa 1 was not detected. In addition, cluster analysis showed that T. gondii strains within type II and III lineage have close relation with strains previously isolated from stray cats in Izmir. Further studies are required to isolate more strains from human cases, other intermediate hosts, and water sources to reveal this relation.

First genetic characterization of Toxoplasma gondii in stray cats from Algeria.

Toxoplasmosis is a parasitic disease with worldwide distribution and a major public health problem. In Algeria, no data are currently available about genotypes of Toxoplasma gondii isolated from animals or humans. The present study assesses for the first time the seroprevalence of toxoplasmosis in stray cats, and provides molecular characterization of T. gondii strains circulating in this feline population in Algiers, the capital city of Algeria. Sera from 96 stray cats were tested for the presence of antibodies against T. gondii using the modified agglutination test. The seroprevalence was 50% (48/96) using 1:6 as the positivity cut-off. Different organs samples from stray cats, including heart samples, were tested for the presence of Toxoplasma DNA using real-time PCR. T. Gondii DNA was detected in 90.6% (87/96) of hearts. Of these parasitic DNAs, 22 were submitted to genotyping through the analysis of 15 microsatellite markers. The identified genotypes (12 of 22) mainly belonged to the type II lineage.

Molecular diagnosis of toxoplasmosis: value of the buffy coat for the detection of circulating Toxoplasma gondii.

Early detection of Toxoplasma tachyzoites circulating in blood using PCR is recommended for immunosuppressed patients at high risk for disseminated toxoplasmosis. Using a toxoplasmosis mouse model, we show that the sensitivity of detection is higher using buffy coat isolated from a large blood volume than using whole blood for this molecular monitoring.

Assembly of the novel five-component apicomplexan multi-aminoacyl-tRNA synthetase complex is driven by the hybrid scaffold protein Tg-p43.

In Toxoplasma gondii, as in other eukaryotes, a subset of the amino-acyl-tRNA synthetases are arranged into an abundant cytoplasmic multi-aminoacyl-tRNA synthetase (MARS) complex. Through a series of genetic pull-down assays, we have identified the enzymes of this complex as: methionyl-, glutaminyl-, glutamyl-, and tyrosyl-tRNA synthetases, and we show that the N-terminal GST-like domain of a partially disordered hybrid scaffold protein, Tg-p43, is sufficient for assembly of the intact complex. Our gel filtration studies revealed significant heterogeneity in the size and composition of isolated MARS complexes. By targeting the tyrosyl-tRNA synthetases subunit, which was found exclusively in the complete 1 MDa complex, we were able to directly visualize MARS particles in the electron microscope. Image analyses of the negative stain data revealed the observed heterogeneity and instability of these complexes to be driven by the intrinsic flexibility of the domain arrangements within the MARS complex. These studies provide unique insights into the assembly of these ubiquitous but poorly understood eukaryotic complexes.

Human toxoplasmosis: which biological diagnostic tests are best suited to which clinical situations?

The Toxoplasma gondii parasite is a worldwide threat most particularly in fetal life and immunosuppression. In most clinical situations (except in some ocular cases), correct detection or identification of toxoplasmosis requires biological analysis. This article considers the laboratory tools that have been developed in this field since the discovery of the pathogen, with emphasis on the most recent tests and how they can or should be used in different clinical situations. The authors also discuss the requirements and pitfalls that one should be aware of when biologically investigating this intriguing parasitosis.

Comparison of the Vidas system and two recent fully automated assays for diagnosis and follow-up of toxoplasmosis in pregnant women and newborns.

Serological testing to detect toxoplasmosis is of major importance to avoid the possible effects of the disease in newborns. This study assessed anti-Toxoplasma IgG and IgM with the Vidas (bioMérieux), Architect (Abbott), and Liaison (DiaSorin) systems in 631 sera from pregnant women and newborns as well as anti-Toxoplasma IgG avidity with these three systems on 54 sera from pregnant women with positive IgG and IgM. The IgG and IgM results were in agreement in, respectively, 95.2% and 98.3% (Vidas versus Architect) and 96.9% and 95.3% (Vidas versus Liaison) of the samples. Specificities were excellent for all the assays, while Vidas sensitivities ranged (depending on the classification of gray zone results) from 93.8 to 98.4% for IgG (Architect, 84.4 to 93.8%; Liaison, 93.8%) and from 81.8 to 90.9% for IgM (Architect, 63.6%; Liaison, 81.8 to 90.9%). In seroconversion sequences, IgMs were generally detected simultaneously by the three assays, while Architect was the earliest assay to detect IgG. In noninfected children, maternally transmitted IgGs were detected for a longer time with Architect than with the other systems. IgMs were positive in only one infected child with the Vidas and Liaison systems. Significantly more sera were classified in the high-avidity category with Vidas than with Architect. This evaluation shows similar performances for Vidas and more recent systems. The Vidas system adequately detects toxoplasmosis in pregnant women and newborns. This system fits the needs of laboratories working on small routine series for first-line testing as well as expert laboratories, due to a high specificity and a powerful avidity test.

Evaluation of the new Elecsys Toxo IgG avidity assay for toxoplasmosis and new insights into the interpretation of avidity results.

Detection and treatment of acute toxoplasmosis during pregnancy can avoid severe disease of the fetus. In this context, assessment of anti-Toxoplasma IgG avidity has been shown to exclude recent infection. The Elecsys Toxo IgG and IgM assays (Roche Diagnostics) have been validated for screening pregnant women and a new assay, Elecsys Toxo IgG Avidity, was recently developed. Our aims were to investigate the performance characteristics of this new avidity assay and explore whether additional information can be provided by avidity assays. The Elecsys assay was compared with the Vidas (bioMérieux) and Architect (Abbott) Avidity assays using two sets of serum samples (n = 291 and n = 255). The rate of general agreement between the Elecsys and Vidas assays was 74%, and that between the Elecsys and Architect assays was 83%. For 11% of the serum samples, avidity was high with the Vidas assay and within the gray zone with the Elecsys assay. None of the assays detected high-avidity antibodies in serum taken <4 months after infection. Avidity values of >90% were exclusively reported in sera taken >9 months after infection by the Elecsys and Architect assays. Almost all avidities of <19% with the Elecsys assay and <17% with the Architect assay corresponded to sera taken <3 and <2 months after infection, respectively. The Elecsys IgG Avidity assay can be used to exclude recent infection. New ways of interpreting the avidity result are also suggested: very high or low values could exclude infections within the last 9 months or help to confirm a recent infection, respectively. However, these potential interpretations require further investigation.

The placenta: a main role in congenital toxoplasmosis?

Systemic infections, such as toxoplasmosis, acquired during pregnancy can lead to placental infection and have profound effects on the mother-to-child relationship and the success of pregnancy. Placental permeability to Toxoplasma gondii is a main parameter that determines parasite transmission to the foetus, and the use of antibiotics to decrease placental parasite load and prevent congenital toxoplasmosis has been suggested for decades. Although parasitological examination of the placenta at birth is commonly used to diagnose neonatal congenital toxoplasmosis, this approach can be controversial. Here we argue in favour of placental examination for both diagnostic and epidemiological purposes.

Detection of Aspergillus fumigatus by quantitative polymerase chain reaction in air samples impacted on low-melt agar.

BACKGROUND: The standard procedure for routine environmental sampling for the prevention of invasive aspergillosis outbreaks is culturing of Aspergillus fumigatus after impaction of air. Time to results is usually 7 days. A preliminary study was carried out to compare the time to results and sensitivity of culturing and quantitative polymerase chain reaction (QPCR) in the detection of airborne A fumigatus. METHODS: Fungal DNA was extracted from 43 samples of impacted low-melt agar by a 3-step extraction method and amplified by QPCR. Identification was made using a specific A fumigatus probe. RESULTS: With QPCR, 19 of the 43 samples were positive for A fumigatus; with culturing, 7 of these 19 samples were positive, and 12 were negative. The cycle threshold (Ct) values for the 12 culture-negative samples were between 39 and 43 cycles, and the Ct values for 6 of the 7 culture-positive samples were <38 cycles, suggesting that the amount of DNA detected by QPCR was higher in the presence of viable conidia. CONCLUSION: QPCR detection of airborne A fumigatus in impacted low-melt agar significantly reduces the period of time between sample collection and results (48 hours), suggesting that this new approach can be beneficial for routine environmental sampling.