Multiomics analysis reveals B. MO1 as a distinct Babesia species and provides insights into its evolution and virulence.

Babesiosis, caused by protozoan parasites of the genus Babesia , is an emerging tick-borne disease of significance for both human and animal health. Babesia parasites infect erythrocytes of vertebrate hosts where they develop and multiply rapidly to cause the pathological symptoms associated with the disease. The identification of various Babesia species underscores the ongoing risk of new zoonotic pathogens capable of infecting humans, a concern amplified by anthropogenic activities and environmental shifts impacting the distribution and transmission dynamics of parasites, their vectors, and reservoir hosts. One such species, Babesia MO1, previously implicated in severe cases of human babesiosis in the midwestern United States, was initially considered closely related to B. divergens , the predominant agent of human babesiosis in Europe. Yet, uncertainties persist regarding whether these pathogens represent distinct variants of the same species or are entirely separate species. We show that although both B. MO1 and B. divergens share similar genome sizes, comprising three nuclear chromosomes, one linear mitochondrial chromosome, and one circular apicoplast chromosome, major differences exist in terms of genomic sequence divergence, gene functions, transcription profiles, replication rates and susceptibility to antiparasitic drugs. Furthermore, both pathogens have evolved distinct classes of multigene families, crucial for their pathogenicity and adaptation to specific mammalian hosts. Leveraging genomic information for B. MO1, B. divergens , and other members of the Babesiidae family within Apicomplexa provides valuable insights into the evolution, diversity, and virulence of these parasites. This knowledge serves as a critical tool in preemptively addressing the emergence and rapid transmission of more virulent strains.

Retrospective study of the epidemiological risk and serological diagnosis of human babesiosis in Asturias, Northwestern Spain.

BACKGROUND: Babesiosis is a globally growing tick-borne disease in humans. Severe babesiosis caused by Babesia divergens has been reported in two patients from Asturias (Northwestern Spain), suggesting an undetected risk for the disease. To analyze this risk, we retrospectively evaluated the seroprevalence of babesiosis in the Asturian population from 2015 through 2017, a period covering the intermediate years in which these two severe cases occurred. METHODS: Indirect fluorescent assay (IFA) and Western blot (WB) were performed to detect B. divergens IgG antibodies in 120 serum samples from Asturian patients infected with the tick-transmitted spirochete Borrelia burgdorferi sensu lato, a condition that indicates exposure to tick bites. RESULTS: This retrospective study confirmed a B. divergens seroprevalence rate of 39.2% according to IFA results. B. divergens incidence was 7.14 cases/100,000 population, exceeding previously reported seroprevalence rates. No differences in epidemiology and risk factors were found between patients infected solely with B. burgdorferi s.l. and those infected with B. burgdorferi s.l. and with IgG antibodies against B. divergens. This last group of patients lived in Central Asturias, had a milder clinical course and, according to WB results, developed different humoral responses against B. divergens. CONCLUSIONS: Babesia divergens parasites have circulated for several years in Asturias. Epidemiological evidence of babesiosis makes Asturias an emerging risk area for this zoonosis. Human babesiosis could also be relevant in other Spanish and European regions affected by borreliosis. Hence, the potential risk of babesiosis on human health in Asturias and other European forest regions needs to be addressed by the health authorities.

Babesia duncani multi-omics identifies virulence factors and drug targets.

Babesiosis is a malaria-like disease in humans and animals that is caused by Babesia species, which are tick-transmitted apicomplexan pathogens. Babesia duncani causes severe to lethal infection in humans, but despite the risk that this parasite poses as an emerging pathogen, little is known about its biology, metabolic requirements or pathogenesis. Unlike other apicomplexan parasites that infect red blood cells, B. duncani can be continuously cultured in vitro in human erythrocytes and can infect mice resulting in fulminant babesiosis and death. We report comprehensive, detailed molecular, genomic, transcriptomic and epigenetic analyses to gain insights into the biology of B. duncani. We completed the assembly, 3D structure and annotation of its nuclear genome, and analysed its transcriptomic and epigenetics profiles during its asexual life cycle stages in human erythrocytes. We used RNA-seq data to produce an atlas of parasite metabolism during its intraerythrocytic life cycle. Characterization of the B. duncani genome, epigenome and transcriptome identified classes of candidate virulence factors, antigens for diagnosis of active infection and several attractive drug targets. Furthermore, metabolic reconstitutions from genome annotation and in vitro efficacy studies identified antifolates, pyrimethamine and WR-99210 as potent inhibitors of B. duncani to establish a pipeline of small molecules that could be developed as effective therapies for the treatment of human babesiosis.

Dynamics of gliotoxin and bis(methylthio)gliotoxin production during the course of Aspergillus fumigatus infection.

As recently described, fungal secondary metabolism activates during infection in response to a hostile host environment. Gliotoxin and bis(methylthio)gliotoxin are two recognized secondary metabolites produced by Aspergillus fumigatus with differential cytotoxicity and involved in virulence. We sought to describe the temporal dynamics of gliotoxin and bis(methylthio)gliotoxin during A. fumigatus progression to further explore their role in the infection. First, we optimized the production of the mycotoxins under different in vitro growth conditions and then specifically measured them using an UHPLC/PDA method. The analytical conditions were selected after testing different parameters such as extraction procedures, column type, and mobile phase composition. We found that gliotoxin and bis(methylthio)gliotoxin are differentially excreted to the extracellular media during the course of A. fumigatus infection regardless of the growth format tested. Dynamic profiles show an early production of gliotoxin, which, after reaching a maximum, decreases coinciding with the increase in the production of the inactive derivative bis(methylthio)gliotoxin. Presence of gliotoxin may indicate an early phase of fungal development, whereas detection of bis(methylthio)gliotoxin may correspond to a more advanced stage of infection. Our chromatographic method successfully characterizes these secondary metabolites. Thus, it may potentially be used to further understand Aspergillus infection. LAY SUMMARY: Aspergillus fumigatus secondary metabolites may contribute to fungal survival. A new chromatographic method was applied to simultaneously characterize two relevant metabolites. Presence of toxic gliotoxin may indicate an early phase of development, whereas the detection of the inactive derivate may represent an advanced infection stage.

Babesia and Human Babesiosis.

Babesia is a genus of intraerythrocytic protozoan parasites belonging to the exclusively parasitic phylum Apicomplexa [...].

Babesia and Theileria Identification in Adult Ixodid Ticks from Tapada Nature Reserve, Portugal.

This study, conducted in a nature reserve in southern Portugal, investigated the frequency and diversity of tick-borne piroplasms in six species of adult ixodid ticks removed from 71 fallow deer (Dama dama) and 12 red deer (Cervus elaphus), collected over the period 2012-2019. The majority of 520 ticks were Ixodes ricinus (78.5%), followed by Rhipicephalus sanguineus sensu lato, Hyalomma lusitanicum, Haemaphysalis punctata, Dermacentor marginatus, and Ixodes hexagonus. The R. sanguineus ticks collected from the deer were clearly exophilic, in contrast to the endophilic species usually associated with dogs. Four tick-borne piroplasms, including Theileria spp., and the zoonotic species, Babesia divergens and Babesia microti, were detected. B. divergens 18S rDNA, identical to that of the bovine reference strain U16370 and to certain strains from red deer, was detected in I. ricinus ticks removed from fallow deer. The sporadic detection of infections in ticks removed from the same individual hosts suggests that the piroplasms were present in the ticks rather than the hosts. Theileria sp. OT3 was found in I. ricinus and, along with T. capreoli, was also detected in some of the other tick species. The natural vector and pathogenic significance of this piroplasm are unknown.

Percutaneous tracheostomy in COVID patients. Experience in our hospital center after one year of pandemic and review of the literature

Background: The pandemic caused by SARS-COV-2 has caused an increase in the need of tracheostomies in patients affected with respiratory distress syndrome. In this article we report our experience during a year of pandemic, we develop our surgical technique to perform percutaneous tracheostomy with the patient in apnea and we compare our results with those of other centers through a bibliographic review. Material and Methods: A one-year retrospective clinical study was carried out on tracheotomies performed on patients admitted to the intensive care unit with severe SARS-CoV-2, with difficulty for ventilation or weaning. The technique performed was percutaneous, with fibroscopic control through the endotracheal tube, keeping the patient under apnea during the opening of the airway, reducing by this method the risk of exposure to the virus. Results: From 35 percutaneous tracheotomies performed, 31% of the patients died from respiratory complications due to SARS-COV-2, but none due to the surgical procedure. The most frequent complication (8.5% of patients) was bleeding around the tracheostoma, resolved with local measures. No healthcare provider involved in the performance of the technique had symptoms or was diagnosed with COVID-19. Conclusions: Our technique of performing percutaneous tracheostomy maintaining apnea during the procedure, under fibroscopic control, has proven to be safe for all those involved in the procedure, and for the patient.(AU)

Integration of Genomic and Transcriptomic Data to Elucidate Molecular Processes in Babesia divergens.

Emerging pathogens have developed ingenious life cycles to facilitate their growth and survival in the host organism. Detailed knowledge of the life cycle of these pathogens is increasingly necessary if we are to design new strategies to prevent infection and transmission. Multi-omics platforms provide useful data at different biological levels, and integration of these data into current approaches can facilitate holistic assessment of emerging pathogens. In this chapter, we bring together various methods and apply an integrative approach for analysis of genomic and transcriptomic data in Babesia divergens, an Apicomplexa emerging parasite that invades red blood cells and causes redwater fever in cattle and the most severe form of babesiosis in humans in Europe. The integrative methodology described herein can be helpful to identify genes active at specific points during life cycle of Apicomplexa parasites.

Integration of Functional Genomic, Transcriptomic, and Metabolomic Data to Identify Key Features in Genomic Expression, Metabolites, and Metabolic Pathways of Babesia divergens.

Upon invasion of red blood cells (RBCs), the Apicomplexa parasite Babesia divergens remains within the RBC for several hours and reproduces asexually, resulting in infective free merozoites that egress and destroy the host cell. Free merozoites rapidly seek and invade new uninfected RBCs. This repetitive cycle allows B. divergens to build a complex population of intraerythrocytic and extracellular stages in the bloodstream of humans and cattle, thus causing babesiosis. To compare biological aspects between B. divergens stages, including the different nature of their metabolism, could be key to our understanding of pathogenesis. Thus, we are currently assessing differences in the B. divergens metabolism of intra- and extracellular (free merozoites) life stages by the use of an integrative approach combining functional genomic, transcriptomic, differential expression, and metabolomic data acquired from sequencing and various analytical platforms. To our knowledge, this is the first effort to describe, in detail, the experimental procedures and integration of different omics to explore the regulation of the metabolism, invasion and proliferation mechanisms of B. divergens. This integrative approach can be used as a reference to study other Apicomplexa parasites.

Tracking the Antibody Immunome in Sporadic Colorectal Cancer by Using Antigen Self-Assembled Protein Arrays.

Sporadic Colorectal Cancer (sCRC) is the third leading cause of cancer death in the Western world, and the sCRC patients presenting with synchronic metastasis have the poorest prognosis. Genetic alterations accumulated in sCRC tumor cells translate into mutated proteins and/or abnormal protein expression levels, which contribute to the development of sCRC. Then, the tumor-associated proteins (TAAs) might induce the production of auto-antibodies (aAb) via humoral immune response. Here, Nucleic Acid Programmable Protein Arrays (NAPPArray) are employed to identify aAb in plasma samples from a set of 50 sCRC patients compared to seven healthy donors. Our goal was to establish a systematic workflow based on NAPPArray to define differential aAb profiles between healthy individuals and sCRC patients as well as between non-metastatic (n = 38) and metastatic (n = 12) sCRC, in order to gain insight into the role of the humoral immune system in controlling the development and progression of sCRC. Our results showed aAb profile based on 141 TAA including TAAs associated with biological cellular processes altered in genesis and progress of sCRC (e.g., FSCN1, VTI2 and RPS28) that discriminated healthy donors vs. sCRC patients. In addition, the potential capacity of discrimination (between non-metastatic vs. metastatic sCRC) of 7 TAAs (USP5, ML4, MARCKSL1, CKMT1B, HMOX2, VTI2, TP53) have been analyzed individually in an independent cohort of sCRC patients, where two of them (VTI2 and TP53) were validated (AUC ~75%). In turn, these findings provided novel insights into the immunome of sCRC, in combination with transcriptomics profiles and protein antigenicity characterizations, wich might lead to the identification of novel sCRC biomarkers that might be of clinical utility for early diagnosis of the tumor. These results explore the immunomic analysis as potent source for biomarkers with diagnostic and prognostic value in CRC. Additional prospective studies in larger series of patients are required to confirm the clinical utility of these novel sCRC immunomic biomarkers.

Intervención psicoterapéutica grupal en una Unidad de Hospitalización Breve de Adolescentes desde las terapias contextuales / Group contextual psychotherapy in an Adolescent Inpatient Psychiatric Unit

Las Unidades de Hospitalización Breve de Niños y Adolescentes son dispositivos para el tratamiento de menores en situación de descompensación psicopatológica aguda cuyo objetivo principal es la contención y estabilización del paciente. Las intervenciones psicoterapéuticas en este tipo de recursos, así como las características de la propia población infanto-juvenil, poseen una serie de particularidades a tener en cuenta de cara a maximizar el potencial psicoterapéutico del ingreso. En el presente trabajo se describe la experiencia de una intervención psicoterapéutica grupal en la Unidad de Hospitalización Breve de Adolescentes del Hospital Universitario Puerta de Hierro-Majadahonda (Madrid). A lo largo del manuscrito se exponen las características del modelo de intervención, basado en la Terapia de Aceptación y Compromiso y la Psicoterapia Analítica Funcional, así como su integración con otras actividades de la Unidad. Además, se ilustran las principales dinámicas, dificultades y desafíos que implica la intervención en este contexto psicoterapéutico específico. (AU)

Child and Adolescent Inpatient Psychiatric Units are resources for the treatment of acute psychopathological decompensation. Their main objectives are the global care and the stabilization of the patient. Psychotherapeutic interventions in this specific context, as well as the characteristics of the child and adolescent population, have certain particularities that should be addressed in order to maximize the psychotherapeutic effects of the intervention during hospital admission. This paper describes the experience of a group psychotherapeutic intervention in the Adolescent Inpatient Psychiatric Unit of Puerta de Hierro-Majadahonda University Hospital (Madrid). The characteristics of the intervention model, based both on Acceptance and Commitment Therapy and Functional Analytic Psychotherapy, are described throughout this manuscript. Additionally, its integration with other activities of the Unit is detailed. Potential difficulties during the intervention, as well as main dynamics and challenges when working with adolescents in this specific context, are also presented. (AU)

A Growth Modulation Index-Based GEISTRA Score as a New Prognostic Tool for Trabectedin Efficacy in Patients with Advanced Soft Tissue Sarcomas: A Spanish Group for Sarcoma Research (GEIS) Retrospective Study.

The aim of this study was to identify an easily reliable prognostic score that selects the subset of advanced soft tissue sarcoma (ASTS) patients with a higher benefit with trabectedin in terms of time to progression and overall survival. A retrospective series of 357 patients with ASTS treated with trabectedin as second- or further-line in 19 centers across Spain was analyzed. First, it was confirmed that patients with high growth modulation index (GMI > 1.33) were associated with the better clinical outcome. Univariate and multivariate analyses were performed to identify factors associated with a GMI > 1.33. Thus, GEISTRA score was based on metastasis free-interval (MFI ≤ 9.7 months), Karnofsky < 80%, Non L-sarcomas and better response in the previous systemic line. The median GMI was 0.82 (0-69), with 198 patients (55%) with a GMI < 1, 41 (11.5%) with a GMI 1-1.33 and 118 (33.1%) with a GMI > 1.33. The lowest GEISTRA score showed a median of time-to-progression (TTP) and overall survival (OS) of 5.7 and 19.5 months, respectively, whereas it was 1.8 and 3.1 months for TTP and OS, respectively, for the GEISTRA 4 score. This prognostic tool can contribute to better selecting candidates for trabectedin treatment in ASTS.

Seminal Plasma and Serum Afamin Levels Are Associated with Infertility in Men with Oligoasthenoteratozoospermia.

The plasma glycoprotein afamin has been previously identified as an alternative carrier protein for vitamin E in extravascular fluids such as plasma and cerebrospinal, ovarian follicular, and seminal fluids. However, to date, no study has established a relationship between afamin levels and infertility in women or men. The purposes of our study were (i) to assess the level of afamin in serum and seminal fluids in infertile men compared to healthy controls and (ii) to study the association between polymorphisms in afamin genes and male infertility. This observational, prospective study evaluated the afamin levels in serum and seminal fluids from infertile men (n = 39) and compared them to those in healthy controls (n = 30). We studied the association between single-nucleotide polymorphisms (SNPs) in the 5`-untranslated region (5`-UTR) of the afamin gene and infertility and analyzed a total of 1000 base pairs from the untranslated region of the afamin gene. Subjects with low sperm motility and low sperm concentration had higher median seminal afamin (18.9 ± 2.9 ng/mg of proteins) and serum afamin concentrations (24.1 ± 4.0 ng/mg of proteins) than subjects with normal sperm parameters (10.6 ± 1.4 ng/mg of proteins) (p < 0.02) (15.6 ± 1.4 ng/mg of proteins) (p < 0.002). A total of five different polymorphisms were found, including one deletion and four single-nucleotide polymorphisms (SNPs). A new transversion (A/T) (position 4:73481093) was identified in an oligoasthenoteratozoospermic patient and was associated with high levels of afamin in plasma and seminal fluids. The prevalence of this variant in our study in the case homozygous for TT is 0.985 (98.5%), and in the case heterozygous for TA is 0.015 (1.5%). Our results suggest that genetic variations in afamin might be associated with male infertility. These findings could significantly enhance our understanding of the molecular genetic causes of infertility.

Potasio elevado, sin T picuda / High pot but no T

Introducción. La identificación y el tratamiento de pacientes con hiperpotasemia son necesarios para prevenir el desarrollo de arritmias. La pseudohiperpotasemia se debe más comúnmente a la hemólisis de la muestra y a menudo es reconocida por los laboratoristas que posteriormente informan los resultados de las pruebas con advertencias de precaución. Los autores presentan un caso de pseudohiperpotasemia en un paciente con leucemia linfocítica crónica. Reporte de caso: los factores técnicos y el método de transporte son una causa potencial de pseudohiperpotasemia. La pseudohiperpotasemia se ha asociado también con hiperleucoctosis, en poblaciones de pacientes con cáncer, más comúnmente en Leucemia linfocítica crónica en adultos, pero también con leucemia linfoblástica aguda en niños. Esto pone al paciente en riesgo de tratamientos innecesarios y potencialmente peligrosos. Conclusión: Los médicos deben considerar la pseudohiperpotasemia como la causa subyacente de los niveles elevados de potasio en pacientes con leucocitosis maligna que no presentan signos o síntomas de hiperpotasemia sistémica.

Introduction. The identification and treatment of patients with hyperkalemia is necessary to prevent the development of arrhythmias. Pseudohyperkalemia is most commonly due to specimen haemolysis and is often recognised by laboratory scientists who subsequently report test results with cautionary warnings. The authors present a case of pseudohyperkalemia in a patient with chronic lymphocytic leukaemia. Report case: the technical factors and method of transport are a potential cause of pseudohyperkalemia. Pseudohyperkalemia has been associated with hyperleukoctosis, in cancer patient populations, more commonly in CLL in adults, but also acute lymphoblastics leukemia in children. This places the patient at risk of unnecessary and potentially dangerous treatments. Conclusion: Physicians should consider pseudohyperkalemia as the underlying cause of elevated potassium levels in patients with malignant leucocytosis who do not have signs or symptom of systemic hyperkalemia.

Four-Dimensional Characterization of the Babesia divergens Asexual Life Cycle, from the Trophozoite to the Multiparasite Stage.

Babesia is an apicomplexan parasite of significance that causes the disease known as babesiosis in domestic and wild animals and in humans worldwide. Babesia infects vertebrate hosts and reproduces asexually by a form of binary fission within erythrocytes/red blood cells (RBCs), yielding a complex pleomorphic population of intraerythrocytic parasites. Seven of them, clearly visible in human RBCs infected with Babesia divergens, are considered the main forms and named single, double, and quadruple trophozoites, paired and double paired pyriforms, tetrad or Maltese Cross, and multiparasite stage. However, these main intraerythrocytic forms coexist with RBCs infected with transient parasite combinations of unclear origin and development. In fact, little is understood about how Babesia builds this complex population during its asexual life cycle. By combining cryo-soft X-ray tomography and video microscopy, main and transitory parasites were characterized in a native whole cellular context and at nanometric resolution. The architecture and kinetics of the parasite population was observed in detail and provide additional data to the previous B. divergens asexual life cycle model that was built on light microscopy. Importantly, the process of multiplication by binary fission, involving budding, was visualized in live parasites for the first time, revealing that fundamental changes in cell shape and continuous rounds of multiplication occur as the parasites go through their asexual multiplication cycle. A four-dimensional asexual life cycle model was built highlighting the origin of several transient morphological forms that, surprisingly, intersperse in a chronological order between one main stage and the next in the cycle.IMPORTANCE Babesiosis is a disease caused by intraerythrocytic Babesia parasites, which possess many clinical features that are similar to those of malaria. This worldwide disease is increasing in frequency and geographical range and has a significant impact on human and animal health. Babesia divergens is one of the species responsible for human and cattle babesiosis causing death unless treated promptly. When B. divergens infects its vertebrate hosts, it reproduces asexually within red blood cells. During its asexual life cycle, B. divergens builds a population of numerous intraerythrocytic (IE) parasites of difficult interpretation. This complex population is largely unexplored, and we have therefore combined three- and four-dimensional imaging techniques to elucidate the origin, architecture, and kinetics of IE parasites. Unveiling the nature of these parasites has provided a vision of the B. divergens asexual cycle in unprecedented detail and is a key step to develop control strategies against babesiosis.

Real-world evidence of the efficacy and tolerability of trabectedin in patients with advanced soft-tissue sarcoma.

INTRODUCTION: The efficacy and tolerability of trabectedin in patients with soft tissue sarcoma (STS) have been confirmed by various clinical studies involving lipo- and leiomyosarcomas as well as many other subtypes including translocation-related sarcomas. These data have been obtained from randomized phase II and III clinical trials. Studies in real-world clinical practice are necessary to bridge the efficacy-effectiveness gap and complete the body of evidence. Furthermore, reinforcing clinical experience with data from routine clinical practice allows drug management to be optimized and clinical benefits to be maximized. AREAS COVERED: The present review provides the most significant data on the efficacy of trabectedin in real-world studies, and the interpretation of real-world experience with trabectedin, in patients with advanced STS. EXPERT OPINION: Trabectedin has demonstrated durable disease control and an adequate safety profile, indicating it to be a suitable long-term treatment drug associated with a good quality of life. Personalized strategies and individualized objectives are the way forward in the management of STS.

MicroRNA expression in relation with clinical evolution of osteosarcoma.

Osteosarcoma is the most common malignant bone tumor. Early diagnosis remains a major challenge, mainly because of the lack of specific biomarkers. We performed miRNAs expression analysis through qPCR in affected and paired healthy bone derived from osteosarcoma patients. Hierarchical clustering using the top ten miRNAs with differential expression showed two main clusters. One integrated by patients with the presence of metastasis or relapse and the other without these complications. Further pathway enrichment analysis reduced to four main miRNAs, hsa-miR-486-3p, hsa-miR-355-5p, hsa-miR-34a-5p and hsa-miR-1228-3p. Afterwards, we compared patients with and without metastasis, the function enrichment analysis along with review of relevant literature, showed that hsa-miR-93-5p and hsa-miR-28-5p were associated with metastasis development. Our results support the relevance of miRNAs in the pathogenesis of osteosarcoma and contribute with evidence regarding the potential role of miRNAs as potential biomarkers. More studies are needed to define the most informative miRNAs in osteosarcoma patients.

Comparative and functional genomics of the protozoan parasite Babesia divergens highlighting the invasion and egress processes.

Babesiosis is considered an emerging disease because its incidence has significantly increased in the last 30 years, providing evidence of the expanding range of this rare but potentially life-threatening zoonotic disease. Babesia divergens is a causative agent of babesiosis in humans and cattle in Europe. The recently sequenced genome of B. divergens revealed over 3,741 protein coding-genes and the 10.7-Mb high-quality draft become the first reference tool to study the genome structure of B. divergens. Now, by exploiting this sequence data and using new computational tools and assembly strategies, we have significantly improved the quality of the B. divergens genome. The new assembly shows better continuity and has a higher correspondence to B. bovis chromosomes. Moreover, we present a differential expression analysis using RNA sequencing of the two different stages of the asexual lifecycle of B. divergens: the free merozoite capable of invading erythrocytes and the intraerythrocytic parasite stage that remains within the erythrocyte until egress. Comparison of mRNA levels of both stages identified 1,441 differentially expressed genes. From these, around half were upregulated and the other half downregulated in the intraerythrocytic stage. Orthogonal validation by real-time quantitative reverse transcription PCR confirmed the differential expression. A moderately increased expression level of genes, putatively involved in the invasion and egress processes, were revealed in the intraerythrocytic stage compared with the free merozoite. On the basis of these results and in the absence of molecular models of invasion and egress for B. divergens, we have proposed the identified genes as putative molecular players in the invasion and egress processes. Our results contribute to an understanding of key parasitic strategies and pathogenesis and could be a valuable genomic resource to exploit for the design of diagnostic methods, drugs and vaccines to improve the control of babesiosis.