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Purpose: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. Methods: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. Results: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. Conclusion: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

The diagnostic assessment of eosinophilias is complex, requiring a multidisciplinary approach and often involving diagnostic challenges. This work aims for a better understanding of the cytomorphological features, immunophenotype, and biological activity of the eosinophil. Additionally, the concepts of peripheral and bone marrow eosinophilia and their potential causes are reviewed. Finally, the review focuses on the broad differential diagnosis of hematologic diseases that may underlie eosinophilia and how to diagnose them. Among the findings that should raise suspicion of hematologic diseases associated with eosinophilia are the presence of splenomegaly and/or lymphadenopathy or an abnormal blood count. In recent years, with advances in molecular techniques, new hematologic malignancies such as myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions are being defined, where eosinophilia can serve as a guiding sign. In these cases, an accurate diagnosis allows for the use of targeted therapy with an improvement in the quality of life and survival of patients., Eosinophils in peripheral blood account for 0.3–5% of leukocytes, which is equivalent to 0.05–0.5 × 109/L. A count above 0.5 × 109/L is considered to indicate eosinophilia, while a count equal to or above 1.5 × 109/L is defined as hypereosinophilia. In bone marrow aspirate, eosinophilia is considered when eosinophils make up more than 6% of the total nuclear cells. In daily clinical practice, the most common causes of reactive eosinophilia are non-hematologic, whether they are non-neoplastic (allergic diseases, drugs, infections, or immunological diseases) or neoplastic (solid tumors). Eosinophilia that is associated with a hematological malignancy may be reactive or secondary to the production of eosinophilopoietic cytokines, and this is mainly seen in lymphoid neoplasms (Hodgkin lymphoma, mature T-cell neoplasms, lymphocytic variant of hypereosinophilic syndrome, and B-acute lymphoblastic leukemia/lymphoma). Eosinophilia that is associated with a hematological malignancy may also be neoplastic or primary, derived from the malignant clone, usually in myeloid neoplasms or with its origin in stem cells (myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions, acute myeloid leukemia with core binding factor translocations, mastocytosis, myeloproliferative neoplasms, myelodysplastic/myeloproliferative neoplasms, and myelodysplastic neoplasms). There are no concrete data in standardized cytological and cytometric procedures that could predict whether eosinophilia is reactive or clonal. The verification is usually indirect, based on the categorization of the accompanying hematologic malignancy. This review focuses on the broad differential diagnosis of hematological malignancies with eosinophilia.

Toxoplasma gondii is a major cause of reproductive failure in small ruminants. Genotypic diversity of T. gondii strains has been associated with variations in phenotypic traits in in vitro and murine models. However, whether such diversity could influence the outcome of infection in small ruminants remains mostly unexplored. Here, we investigate the outcome of oral challenge in sheep at mid-pregnancy with 10 sporulated oocysts from three different T. gondii isolates belonging to archetypal II and III and selected according to their genetic and phenotypic variations shown in previous studies. Seventy-three pregnant sheep were divided in four groups: G1 infected with TgShSp1 isolate (type II, ToxoDB#3), G2 with TgShSp16 isolate (type II, ToxoDB#3), G3 with TgShSp24 isolate (type III, ToxoDB#2) and G4 of uninfected control sheep. Two different approaches were carried out within this study: (i) the outcome for the pregnancy after infection (n = 33) and (ii) the lesions and parasite tropism and burden at 14 and 28 days post infection (dpi) (n = 40). The onset of hyperthermia and seroconversion occurred one and two days later, respectively in G1 when compared to G2 and G3. However, sheep that suffered from reproductive failure, either by abortion, foetal dead at the time of euthanasia or stillbirth were similar among infected groups (50%, 40% and 47%, respectively). Histological lesions in placentomes and foetal tissues from euthanized animals from the second approach were only detected at 28 dpi and mainly in G1. At 14 dpi, T. gondii-DNA was only detected in G1 in the 11% of the placentomes. However, at 28 dpi the frequency of detection in placentomes was higher in G1 (96%) than in G2 and G3 (7% and 47%, respectively) besides in foetuses was lower in G2 (20%) than in G1 and G3 (100% and 87%, respectively). Regarding late abortions, stillbirths, and lambs of G1, G2 and G3, the frequency of microscopic lesions was similar between groups (79%, 78% and 67%, respectively) whe