Immunoglobulin and T cell receptor repertoire changes induced by a prototype vaccine against Chagas disease in naïve rhesus macaques.

BACKGROUND: A vaccine against Trypanosoma cruzi, the agent of Chagas disease, would be an excellent additional tool for disease control. A recombinant vaccine based on Tc24 and TSA1 parasite antigens was found to be safe and immunogenic in naïve macaques. METHODS: We used RNA-sequencing and performed a transcriptomic analysis of PBMC responses to vaccination of naïve macaques after each vaccine dose, to shed light on the immunogenicity of this vaccine and guide the optimization of doses and formulation. We identified differentially expressed genes and pathways and characterized immunoglobulin and T cell receptor repertoires. RESULTS: RNA-sequencing analysis indicated a clear transcriptomic response of PBMCs after three vaccine doses, with the up-regulation of several immune cell activation pathways and a broad non-polarized immune profile. Analysis of the IgG repertoire showed that it had a rapid turnover with novel IgGs produced following each vaccine dose, while the TCR repertoire presented several persisting clones that were expanded after each vaccine dose. CONCLUSIONS: These data suggest that three vaccine doses may be needed for optimum immunogenicity and support the further evaluation of the protective efficacy of this vaccine.

Monkeypox Virus Evolution before 2022 Outbreak.

Phylogenetic analysis of monkeypox virus genomes showed statistically significant divergence and nascent subclades during the 2022 mpox outbreak. Frequency of G>A/C>T transitions has increased in recent years, probably resulting from apolipoprotein B mRNA editing enzyme catalytic polypeptide 3G (APOBEC3) deaminase editing. This microevolutionary pattern most likely reflects community spread of the virus and adaptation to humans.

Posterior wall ablation by pulsed-field ablation: procedural safety, efficacy, and findings on redo procedures.

AIMS: The left atrial posterior wall is a potential ablation target in patients with recurrent atrial fibrillation despite durable pulmonary vein isolation or in patients with roof-dependent atrial tachycardia (AT). Pulsed-field ablation (PFA) offers efficient and safe posterior wall ablation (PWA), but available data are scarce. METHODS AND RESULTS: Consecutive patients undergoing PWA using PFA were included. Posterior wall ablation was performed using a pentaspline PFA catheter and verified by 3D-electroanatomical mapping. Follow-up was performed using 7-day Holter ECGs 3, 6, and 12 months after ablation. Recurrence of any atrial arrhythmia lasting more than 30 s was defined as failure. Lesion durability was assessed during redo procedures. Posterior wall ablation was performed in 215 patients (70% males, median age 70 [IQR 61-75] years, 67% redo procedures) and was successful in all patients (100%) by applying a median of 36 (IQR 32-44) PFA lesions. Severe adverse events were cardiac tamponade and vascular access complication in one patient each (0.9%). Median follow-up was 7.3 (IQR 5.0-11.8) months. One-year arrhythmia-free outcome in Kaplan-Meier analysis was 53%. A redo procedure was performed in 26 patients (12%) after a median of 6.9 (IQR 2.4-11) months and showed durable PWA in 22 patients (85%) with only minor lesion regression. Among four patients with posterior wall reconnection, three (75%) presented with roof-dependent AT. CONCLUSION: Posterior wall ablation with this pentaspline PFA catheter can be safely and efficiently performed with a high durability observed during redo procedures. The added value of durable PWA for the treatment of atrial fibrillation remains to be evaluated.

Genetic diversity of Trypanosoma cruzi infecting raccoons (Procyon lotor) in 2 metropolitan areas of southern Louisiana: implications for parasite transmission networks.

Trypanosoma cruzi, the aetiological agent of Chagas disease, exists as an anthropozoonosis in Louisiana. Raccoons are an important reservoir, as they demonstrate high prevalence and maintain high parasitaemia longer than other mammals. Given the complex nature of parasite transmission networks and importance of raccoons as reservoirs that move between sylvatic and domestic environments, detailing the genetic diversity of T. cruzi in raccoons is crucial to assess risk to human health. Using a next-generation sequencing approach targeting the mini-exon, parasite diversity was assessed in 2 metropolitan areas of Louisiana. Sequences were analysed along with those previously identified in other mammals and vectors to determine if any association exists between ecoregion and parasite diversity. Parasites were identified from discrete typing units (DTUs) TcI, TcII, TcIV, TcV and TcVI. DTUs TcII, TcV and TcVI are previously unreported in raccoons in the United States (US). TcI was the most abundant DTU, comprising nearly 80% of all sequences. All but 1 raccoon harboured multiple haplotypes, some demonstrating mixed infections of different DTUs. Furthermore, there is significant association between DTU distribution and level III ecoregion in Louisiana. Finally, while certain sequences were distributed across multiple tissues, others appeared to have tissue-specific tropism. Taken together, these findings indicate that ongoing surveillance of T. cruzi in the US should be undertaken across ecoregions to fully assess risk to human health. Given potential connections between parasite diversity and clinical outcomes, deep sequencing technologies are crucial and interventions targeting raccoons may prove useful in mitigating human health risk.

[Epicardial adipose tissue and atrial fibrillation]. / Tissu adipeux épicardique et fibrillation auriculaire.

Atrial fibrillation (AF) is the most common arrhythmia encountered in adults; it is associated with a significant morbidity and mortality. Obesity is a risk factor contributing to AF occurrence. Recently, interest has focused on epicardial adipose tissue (EAT), defined as a fatty deposit located between the epicardium and the visceral pericardium. Its characteristics are distinct from classic adipose deposits: it infiltrates the epicardial myocardium and secretes cytokines, which modulate cardiomyocyte electrophysiology and cardiac remodeling. Different studies show that EAT can be an independent risk factor for AF and that EAT thickness, as measured by CT or MRI, could predict the presence, severity and recurrence of AF.

La fibrillation auriculaire (FA) est l'arythmie la plus fréquemment rencontrée chez l'adulte ; elle est associée à une morbi-mortalité importante. L'obésité est un facteur de risque contribuant à sa survenue. Récemment, l'intérêt s'est porté sur le tissu adipeux épicardique (TAE), défini comme un dépôt adipeux situé entre l'épicarde et le péricarde viscéral. Ses caractéristiques sont distinctes des dépôts adipeux classiques : il infiltre le myocarde épicardique et sécrète des cytokines modulant l'électrophysiologie des cardiomyocytes et provoquant un remodelage fibro-adipeux cardiaque. Différentes études montrent que le TAE peut être un facteur de risque indépendant de survenue de FA. L'épaisseur du TAE mesurée par CT-scan ou par IRM pourrait être utilisée comme facteur prédictif de la présence, de la gravité et de la récidive de FA.

Geographic Variations in Test Reactivity for the Serological Diagnosis of Trypanosoma cruzi Infection.

Chagas disease is a neglected disease caused by Trypanosoma cruzi parasites. Most diagnosis is based on serological tests, but the lack of a gold standard test complicates the measurement of test performance. To overcome this limitation, we used samples from a cohort of well-characterized T. cruzi-infected women to evaluate the reactivity of two rapid diagnostic tests and one enzyme-linked immunosorbent assay (ELISA). Our cohort was derived from a previous study on congenital transmission of T. cruzi and consisted of 481 blood/plasma samples from Argentina (n = 149), Honduras (n = 228), and Mexico (n = 104), with at least one positive T. cruzi PCR. Reactivity of the three tests ranged from 70.5% for the Wiener ELISA to 81.0% for the T-Detect and 90.4% for the Stat-Pak rapid tests. Test reactivity varied significantly among countries and was highest in Argentina and lowest in Mexico. When considering at least two reactive serological tests to confirm seropositivity, over 12% of T. cruzi infection cases from Argentina were missed by serological tests, over 21% in Honduras, and an alarming 72% in Mexico. Differences in test performance among countries were not due to differences in parasitemia, but differences in antibody levels against ELISA antigens were observed. Geographic differences in T. cruzi parasite strains as well as genetic differences among human populations both may contribute to the discrepancies in serological testing. Improvements in serological diagnostics for T. cruzi infections are critically needed to ensure an optimum identification of cases.

Sequence of Trypanosoma cruzi reference strain SC43 nuclear genome and kinetoplast maxicircle confirms a strong genetic structure among closely related parasite discrete typing units.

Chagas disease is a zoonotic, parasitic, vector-borne neglected tropical disease that affects the lives of over 6 million people throughout the Americas. Trypanosoma cruzi, the causative agent, presents extensive genetic diversity. Here we report the genome sequence of reference strain SC43cl1, a hybrid strain belonging to the TcV discrete typing unit (DTU). The assembled diploid genome was 79 Mbp in size, divided into 1236 contigs with an average coverage reaching 180×. There was extensive synteny of SC43cl1 genome with closely related TcV and TcVI genomes, with limited sequence rearrangements. TcVI genomes included several expansions not present in TcV strains. Comparative analysis of both nuclear and kinetoplast sequences clearly separated TcV from TcVI strains, which strongly supports the current DTU classification.

Shelter cats host infections with multiple Trypanosoma cruzi discrete typing units in southern Louisiana.

Trypanosoma cruzi is a zoonotic parasite endemic in the southern US and the Americas, which may frequently infect dogs, but limited information is available about infections in cats. We surveyed a convenience sample of 284 shelter cats from Southern Louisiana to evaluate T. cruzi infection using serological and PCR tests. Parasites from PCR positive cats were also genotyped by PCR and deep sequencing to assess their genetic diversity. We detected a seropositivity rate for T. cruzi of at least 7.3% (17/234), and 24.6% of cats (70/284) were PCR positive for the parasite. Seropositivity increased with cat age (R2 = 0.91, P = 0.011), corresponding to an incidence of 7.2% ± 1.3 per year, while PCR positivity decreased with age (R2 = 0.93, P = 0.007). Cats were predominantly infected with parasites from TcI and TcVI DTUs, and to a lesser extent from TcIV and TcV DTUs, in agreement with the circulation of these parasite DTUs in local transmission cycles. These results indicate that veterinarians should have a greater awareness of T. cruzi infection in pets and that it would be important to better evaluate the risk for spillover infections in humans.

Prevalence and risk factors for vitamin D deficiency in long-term childhood cancer survivors.

BACKGROUND: Childhood cancer survivors (CCS) have increased risk of developing chronic health conditions, including musculoskeletal disorders. Little is known regarding vitamin D deficiency (VDD, <20 ng/ml) and its association with bone mineral density (BMD) in long-term CCS. We evaluated the prevalence and risk factors for VDD in a large, diverse population of long-term CCS, and examined the association between VDD and BMD in patients who underwent guideline-recommended dual-energy X-ray absorptiometry (DXA) screening. METHODS: This cross-sectional study included 446 consecutive CCS seen from March 2018 to September 2020. Univariate analyses examined associations between CCS demographics, socioeconomic status, and treatment exposures and VDD. Multivariable logistic regressions identified factors associated with odds of VDD and reduced BMD. RESULTS: Median age at evaluation was 27.5 years (range 7-67 years); median time from completing therapy was 14.2 years (range 2-65 years). Fifty percent were female, and 45% were Hispanic. Twenty-four percent had VDD. In multivariable analysis, overweight and obese BMI were associated with VDD (overweight: OR 1.78, 95% CI 1.03-3.07, p = 0.04; obese: OR 2.40, 95% CI 1.39-4.13, p < 0.01; reference: normal/underweight), as was Hispanic or black race/ethnicity (OR 2.40, 95% CI 1.41-4.09, p < 0.01; reference: non-Hispanic white). In the 118 CCS with DXA results, VDD was independently associated with reduced BMD (OR 3.58, 95%CI 1.33-9.59, p = 0.01). CONCLUSIONS: CCS have a high prevalence of VDD. High BMI and Hispanic or black race/ethnicity were associated with VDD. Survivors with VDD had a greater than threefold risk of reduced BMD. Risk-based screening may facilitate timely interventions to mitigate VDD and improve BMD in CCS.

Dry and Rainy Seasons Significantly Alter the Gut Microbiome Composition and Reveal a Key Enterococcus sp. (Lactobacillales: Enterococcaceae) Core Component in Spodoptera frugiperda (Lepidoptera: Noctuidae) Corn Strain From Northwestern Colombia.

Spodoptera frugiperda is a polyphagous pest of several crops of economic importance. Nowadays, the insect is broadly distributed in America and, recently, in Africa, Asia, and Australia. The species has diverged into corn and rice strains. The role of the gut microbiota in insect physiology is relevant due to its participation in crucial functions. However, knowledge of seasonal variations that alter the gut microbiome in pests is limited. Gut microbiome composition between the dry and rainy seasons was analyzed with cultured and uncultured approaches in S. frugiperda corn strain larvae collected at Northwest Colombia, as seasonal microbiome changes might fluctuate due to environmental changes. On the basis of culture-dependent methods, results show well-defined microbiota with bacterial isolates belonging to Enterococcus, Klebsiella (Enterobacteriales: Enterobacteriaceae), Enterobacter (Enterobacterales: Enterobacteriaceae), and Bacillus (Bacillales: Bacillaceae) genera. The community composition displayed a low bacterial diversity across all samples. The core community detected with uncultured methods was composed of Enterococcus, Erysipelatoclostridium (Erysipelotrichales: Erysipelotrichaceae), Rasltonia (Burkholderiales: Burkholderiaceae), and Rhizobium (Hyphomicrobiales: Rhizobiaceae) genera, and Enterobacteriaceae family members. Significant differences in microbiome diversity were observed between the two seasons. The relative abundance of Erysipelatoclostridium was high in the dry season, while in the phylotype ZOR0006 (Erysipelotrichales: Erysipelotrichaceae) and Tyzzerella (Lachnospirales: Lachnospiraceae) genus, the relative abundance was high in the rainy season. The overall low gut bacterial diversity observed in the S. frugiperda corn strain suggests a strong presence of antagonist activity as a selection factor possibly arising from the host, the dominant bacterial types, or the material ingested. Targeting the stability and predominance of this core microbiome could be an additional alternative to pest control strategies, particularly in this moth.

Two New Strains of Wolbachia Affecting Natural Avocado Thrips.

Wolbachia is an obligate intracellular bacterium with a high frequency of infection and a continental distribution in arthropods and nematodes. This endosymbiont can induce various reproductive phenotypes in their hosts and has been previously found naturally in several pests including thrips (Thripidae). These insects cause physical fruit damage and economic losses in avocado. The presence of Wolbachia was evaluated for the first time in avocado thrips populations of Frankliniella sp. and Scirtothrips hansoni sp.n. from eastern Antioquia. DNA from adult thrips individuals was used to assess the detection of Wolbachia by amplifying a fragment (600 bp) of the Wolbachia major surface protein (wsp) gene. Results confirmed the presence of two new Wolbachia strains in these two thrips species, with a higher percentage of natural infection in S. hansoni sp.n. The first Wolbachia species was found in Frankliniella sp. and belongs to supergroup A and the second was detected in S. hansoni sp.n. and is part of supergroup B. Wolbachia was more frequently found in females (32.73%), and only found in one male. Analysis of phylogenetic relationships, suggests that the two new Wolbachia sequences (wFran: Frankliniella and wShan: Scirtothrips hansoni) detected here represent two new groups for this endosymbiont. The haplotype network shows the presence of two possible haplotypes for each strain. Future studies to evaluate the possible use of Wolbachia as a control agent in avocado thrips are necessary. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00951-5.

Interactions among Triatoma sanguisuga blood feeding sources, gut microbiota and Trypanosoma cruzi diversity in southern Louisiana.

Integrating how biodiversity and infectious disease dynamics are linked at multiple levels and scales is highly challenging. Chagas disease is a vector-borne disease, with specificities of the triatomine vectors and Trypanosoma cruzi parasite life histories resulting in a complex multihost and multistrain life cycle. Here, we tested the hypothesis that T. cruzi transmission cycles are shaped by triatomine host communities and gut microbiota composition by comparing the integrated interactions of Triatoma sanguisuga in southern Louisiana with feeding hosts, T. cruzi parasite and bacterial microbiota in two habitats. Bugs were collected from resident's houses and animal shelters and analysed for genetic structure, blood feeding sources, T. cruzi parasites, and bacterial diversity by PCR amplification of specific DNA markers followed by next-generation sequencing, in an integrative metabarcoding approach. T. sanguisuga feeding host communities appeared opportunistic and defined by host abundance in each habitat, yielding distinct parasite transmission networks among hosts. The circulation of a large diversity of T. cruzi DTUs was also detected, with TcII and TcV detected for the first time in triatomines in the US. The bacterial microbiota was highly diverse and varied significantly according to the DTU infecting the bugs, indicating specific interactions among them in the gut. Expanding such studies to multiple habitats and additional triatomine species would be key to further refine our understanding of the complex life cycles of multihost, multistrain parasites such as T. cruzi, and may lead to improved disease control strategies.

Short-course Benznidazole treatment to reduce Trypanosoma cruzi parasitic load in women of reproductive age (BETTY): a non-inferiority randomized controlled trial study protocol.

BACKGROUND: Retrospective observational studies suggest that transmission of Trypanosoma cruzi does not occur in treated women when pregnant later in life. The level of parasitemia is a known risk factor for congenital transmission. Benznidazole (BZN) is the drug of choice for preconceptional treatment to reduce parasitic load. The fear of treatment-related side effects limits the implementation of the Argentine guideline recommending BZN 60d/300 mg (or equivalent) treatment of T. cruzi seropositive women during the postpartum period to prevent transmission in a future pregnancy. A short and low dose BZN treatment might reduce major side effects and increase compliance, but its efficacy to reduce T. cruzi parasitic load compared to the standard 60d/300 mg course is not yet established. Clinical trials testing alternative BZN courses among women of reproductive age are urgently needed. METHODS AND DESIGN: We are proposing to perform a double-blinded, non-inferiority randomized controlled trial comparing a short low dose 30-day treatment with BZN 150 mg/day (30d/150 mg) vs. BZN 60d/300 mg. We will recruit not previously treated T. cruzi seropositive women with a live birth during the postpartum period in Argentina, randomize them at 6 months postpartum, and follow them up with the following specific aims: Specific aim 1: to measure the effect of BZN 30d/150 mg compared to 60d/300 mg preconceptional treatment on parasitic load measured by the frequency of positive Polymerase Chain Reaction (PCR) (primary outcome) and by real-time quantitative PCR (qPCR), immediately and 10 months after treatment. Specific aim 2: to measure the frequency of serious adverse events and/or any adverse event leading to treatment interruption. TRIAL REGISTRATION: ClinicalTrials.gov . Identifier: NCT03672487 . Registered 14 September 2018.

Molecular Genotyping of Trypanosoma cruzi by Next-Generation Sequencing of the Mini-Exon Gene Reveals Infections With Multiple Parasite Discrete Typing Units in Chagasic Patients From Yucatan, Mexico.

The diversity of Trypanosoma cruzi parasites infecting humans is still poorly understood. We used deep sequencing to analyze this diversity in chagasic patients from Mexico. Such information is crucial to understand transmission cycles and to identify determinants of epidemiological and clinical characteristics of the infection. We analyzed parasite mini-exon spliced-leader sequences following amplification of blood DNA by polymerase chain reaction and deep sequencing. Chagasic patients presented a diverse assemblage of parasite haplotypes covering TcI, TcII, TcV, and TcVI discrete typing units, with a mean (±SEM) of 3.9 ± 0.7 haplotypes/patient, and 47% harbored infections with multiple discrete typing units. Most parasite haplotypes from patients were identical or similar to those for Triatoma dimidiata from the same region, confirming their local circulation. Infection with multiple T. cruzi strains may influence serological diagnostic test results and disease progression in patients and should be taken into account to evaluate associations between parasite diversity and clinical aspects of T. cruzi infections.

Granulocyte-monocyte apheresis: an alternative combination therapy after loss of response to anti-TNF agents in ulcerative colitis.

Objective: To evaluate the effectiveness and safety of the combination of granulocyte-monocyte apheresis (GMA) after loss of response (LOR) to anti-tumor necrosis factor (TNF) agents in ulcerative colitis (UC). Materials and methods: A retrospective, multicenter study was performed in 11 inflammatory bowel disease (IBD) Units. Clinical remission was defined as a partial Mayo score ≤2. The effectiveness of the treatment was evaluated by the partial Mayo score and the rate of anti-TNF intensification, switch, swap or colectomy. Results: Forty-seven patients with ulcerative colitis were included (mean age 35 years, mean disease duration 52 months, 66% male and 59% extensive colitis). Twenty-three subjects were receiving infliximab, eighteen adalimumab and six golimumab. GMA was combined after a primary non-response (49%) or secondary loss of response (51%) to anti-TNF therapy. We observed a significant decrease in partial Mayo score and fecal calprotectin after GMA. Fifteen patients (32%) responded to the combination therapy without anti-TNF intensification, switch, swap or colectomy. Eight patients (17%) underwent colectomy. Two patients (4%) presented adverse events related to the technique. Conclusions: Combination of GMA and anti-tumor necrosis factor is a safe and effective treatment after the loss of response to these biologic agents, with a significant decrease of the clinical disease activity and biomarkers, in a population with limited therapeutic alternatives.

Assessment of human health risk associated with methylmercury in the imported fish marketed in the Caribbean.

The decline in marine and freshwaters catches in recent years in Colombia has led to a change in dietary habits, with an increase in the purchase and consumption of imported fish. This is of particular concern as fish are sometimes caught in mercury-contaminated waters, and are subsequently sold canned or uncanned. In addition, canned tuna has received little attention as it is widely assumed that concentrations are low. In this study, total mercury (THg) and methylmercury (MeHg) concentrations were evaluated in three imported fish species marketed in Colombia, Prochilodus lineatus, Prochilodus reticulatus, and Pangasianodon hypophthalmus, plus four brands of canned tuna and one of sardines. One brand of tuna showed the highest mean concentrations of THg (0.543 ±â€¯0.237 µg/g, wet weight, ww) and MeHg (0.518 ±â€¯0.337 µg/g ww), while concentrations in P. hypophthalmus were approximately 30 times lower (≈0.02 µg/g ww). The estimated weekly intake (EWI) in children was above the provisional tolerable weekly intake (PTWI) of MeHg established by the Joint FAO/World Health Organization (WHO) Expert Committee on Food Additives (JECFA) in 2007, 1.6 µg/kg body weight (bw) per week, for all the canned tuna brands. Values for adults were below PTWI, whereas for women of childbearing age, values were above PTWI only for brand D of canned tuna. The estimate of the potential risk indicated that MeHg levels in canned tuna can generate negative effects in vulnerable groups, while the EWI of fresh fish did not pose a threat to the general population. Therefore, establishing strategies to address the high consumption of canned tuna, and continuous monitoring to control commercial food, are recommended to decrease Hg exposure.

Phytochemical Investigation of Male and Female Hedyosmum scabrum (Ruiz & Pav.) Solms Leaves from Ecuador.

This article reports the chemical composition of the essential oils obtained by hydrodistillation of male and female H. scabrum fresh leaves. The essential oils, HSMO and HSFO, respectively, were analyzed by GC/MS and GC-FID. A total of 93 components were detected, accounting for 94.8% and 95.3% of HSMO and HSFO, respectively. The prevalent constituents of HSMO were pinocarvone (13.1%), d-germacren-4-ol (12.6%), 1,8-cineole (10.8%), α-pinene (6.4%), and ß-pinene (4.8%), whereas the major components of HSFO were 1,8-cineole (20.5%), linalool (16.5%), α-pinene (15.0%), ß-pinene (6.4%), and sabinene (6.3%). The different enantiomeric distribution of ß-pinene, sabinene, limonene, linalool in the two oils, was determined. The non-volatile esters of p-coumaric and ferulic acids with borneol (1 and 4), cis-chrysanthenol (2 and 5), and cis-pinocarveol (3 and 6) were identified in the leaves after basic hydrolysis and analysis of the NMR spectra of the free acids, and GC/MS spectra of the monoterpene alcohols, respectively. Compounds 2, 3, 5, and 6 have been found in nature for the first time. These findings demonstrated that, from a chemical point of view, male and female individuals of H. scabrum collected in Ecuador seem quite differentiated between each other and from samples of the same species growing in Bolivia and in Peru.

Striking Divergence in Toxoplasma ROP16 Nucleotide Sequences From Human and Meat Samples.

BACKGROUND: ROP16 is a protein kinase of Toxoplasma gondii identified in the mouse model as a virulent marker, but it is unknown whether this finding is relevant in human toxoplasmosis. METHODS: We obtained the Toxoplasma ROP16 locus DNA sequence in samples from 12 patients with ocular toxoplasmosis, 1 sample from a patient with congenital toxoplasmosis, 22 samples from soldiers operating in the jungle, 2 samples from urban soldiers, and 10 samples from meat for human consumption. An enzyme-linked immunosorbent assay specific for antibodies against the ROP16 mouse-virulent peptide was performed in 46 serum specimens from patients with ocular toxoplasmosis and in 28 serum specimens from patients with chronic asymptomatic infection, of whom 19 had congenital infection and 11 had toxoplasmic lymphadenitis. RESULTS: We found a striking divergence of the ROP16 nucleotide sequences. Ten of 12 sequences (83.3%) from patients with ocular toxoplasmosis clustered with those of mouse-virulent strains, whereas 7 of 7 ROP16 sequences (100%) from meat were clustered with those of mouse-avirulent strains. Only 11 of 104 serum specimens (10.5%) had specific antibodies against the mouse-virulent peptide, and there was no association between clinical forms and positive results of serological assays. CONCLUSIONS: The majority of ROP16 nucleotide sequences from Colombian patients with ocular toxoplasmosis belonged to the group of mouse-virulent strains.

Trypanosoma cruzi amastigote transcriptome analysis reveals heterogenous populations with replicating and dormant parasites.

Trypanosoma cruzi is a protozoan parasite causing Chagas disease, with a complex life cycle involving different stages in insect vectors and mammalian hosts. Amastigotes are an intracellular form that replicates in the cytoplasm of host cells, and recent studies suggested that dormant forms may be contributing to parasite persistence, suggesting cellular heterogeneity among amastigotes. We investigated here if a transcriptomic approach could identify some heterogeneity in intracellular amastigotes and identify a dormant population. We used gene expression data derived from bulk RNA-sequencing of T. cruzi infection of human fibroblasts for deconvolution using CDSeq, which allows to simultaneously estimate amastigote cell-type proportions and cell-type-specific expression profiles. Six amastigote subpopulations were identified, confirming intracellular amastigotes heterogeneity, and one population presented characteristics of non-replicative dormant parasites, based on replication markers and TcRAD51 expression. Transcriptomic approaches appear to be powerful to understand T. cruzi cell differentiation and expansion of these studies could provide further insight on the role different cell types in parasite persistence and Chagas disease pathogenesis.

Identification of highly conserved Trypanosoma cruzi antigens for the development of a universal serological diagnostic assay.

Chagas Disease is an important neglected tropical disease caused by Trypanosoma cruzi. There is no gold standard for diagnosis and commercial serological tests perform poorly in certain locations. By aligning T. cruzi genomes covering parasite genetic and geographic diversity, we identified highly conserved proteins that could serve as universal antigens for improved diagnosis. Their antigenicity was tested in high-density peptide microarrays using well-characterized plasma samples, including samples presenting true infections but discordant serology. Individual and combination of epitopes were also evaluated in peptide-ELISAs. We identified >1400 highly conserved T. cruzi proteins evaluated in microarrays. Remarkably, T. cruzi positive controls had a different epitope recognition profile compared to serologically discordant samples. In particular, multiple T. cruzi antigens used in current tests and their strain-variants, and novel epitopes thought to be broadly antigenic failed to be recognized by discordant samples. Nonetheless, >2000 epitopes specifically recognized by IgGs from both positive controls and discordant samples were identified. Evaluation of selected peptides in ELISA further illustrated the extensive variation in antibody profiles among subjects and a peptide combination could outperform a commercial ELISA, increasing assay sensitivity from 52.3% to 72.7%. Individual variation in antibody profiles rather than T. cruzi diversity appears to be the main factor driving differences in serological diagnostic performance according to geography, which will be important to further elucidate. ELISA with a combination of peptides recognized by a greater number of individuals could better capture infections, and further development may lead to an optimal antigen mixture for a universal diagnostic assay.