Longitudinal anti-SARS-CoV-2 antibody immune response in acute and convalescent patients.

Despite global efforts to assess the early response and persistence of SARS-CoV-2 antibodies in patients infected with or recovered from COVID-19, our understanding of the factors affecting its dynamics remains limited. This work aimed to evaluate the early and convalescent immunity of outpatients infected with SARS-CoV-2 and to determine the factors that affect the dynamics and persistence of the IgM and IgG antibody response. Seropositivity of volunteers from Mexico City and the State of Mexico, Mexico, was evaluated by ELISA using the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein for 90 days, at different time points (1, 15, 45, 60, and 90 days) after molecular diagnosis (RT-qPCR). Gender, age range, body mass index (BMI), comorbidities, and clinical spectrum of disease were analyzed to determine associations with the dynamics of anti-SARS-CoV-2 antibodies. On 90 days post-infection, individuals with moderate and asymptomatic disease presented the lowest levels of IgM, while for IgG, at the same time, the highest levels occurred with mild and moderate disease. The IgM and IgG levels were related to the clinical spectrum of disease, BMI, and the presence/absence of comorbidities through regression trees. The results suggest that the dynamics of anti-SARS-CoV-2 IgM and IgG antibodies in outpatients could be influenced by the clinical spectrum of the disease. In addition, the persistence of antibodies against SARS-CoV-2 could be related to the clinical spectrum of the disease, BMI, and the presence/absence of comorbidities.

Amoebiasis: Advances in Diagnosis, Treatment, Immunology Features and the Interaction with the Intestinal Ecosystem.

This review of human amoebiasis is based on the most current knowledge of pathogenesis, diagnosis, treatment, and Entamoeba/microbiota interactions. The most relevant findings during this last decade about the Entamoeba parasite and the disease are related to the possibility of culturing trophozoites of different isolates from infected individuals that allowed the characterization of the multiple pathogenic mechanisms of the parasite and the understanding of the host-parasite relationship in the human. Second, the considerable advances in molecular biology and genetics help us to analyze the genome of Entamoeba, their genetic diversity, and the association of specific genotypes with the different amoebic forms of human amoebiasis. Based on this knowledge, culture and/or molecular diagnostic strategies are now available to determine the Entamoeba species and genotype responsible for invasive intestinal or extraintestinal amoebiasis cases. Likewise, the extensive knowledge of the immune response in amoebiasis with the appearance of new technologies made it possible to design diagnostic tools now available worldwide. Finally, the understanding of the interaction between the Entamoeba species and the intestinal microbiota aids the understanding of the ecology of this parasite in the human environment. These relevant findings will be discussed in this review.

The fecal and oropharyngeal eukaryotic viromes of healthy infants during the first year of life are personal.

Using a metagenomic sequencing approach, we described and compared the diversity and dynamics of the oropharyngeal and fecal eukaryotic virome of nine asymptomatic children in a semi-rural community setting located in the State of Morelos, Mexico. Ninety oropharyngeal swabs and 97 fecal samples were collected starting 2 weeks after birth and monthly thereafter until 12 months of age. In both niches, more than 95% of the total sequence reads were represented by viruses that replicate either in humans or in plants. Regarding human viruses, three families were most abundant and frequent in the oropharynx: Herpesviridae, Picornaviridae, and Reoviridae; in fecal samples, four virus families predominated: Caliciviridae, Picornaviridae, Reoviridae, and Anelloviridae. Both niches showed a high abundance of plant viruses of the family Virgaviridae. Differences in the frequency and abundance of sequence reads and diversity of virus species were observed in both niches and throughout the year of study, with some viruses already present in the first months of life. Our results suggest that the children's virome is dynamic and likely shaped by the environment, feeding, and age. Moreover, composition analysis suggests that the virome composition is mostly individual. Whether this constant exposition to different viruses has a long-term impact on children's health or development remains to be studied.

The regulatory function of Blastocystis spp. on the immune inflammatory response in the gut microbiome.

Blastocystis spp. is a unicellular organism that resides in digestive tract of various vertebrates, with a worldwide distribution and a variable prevalence. For many years, Blastocystis spp. was considered a cyst of a flagellate, a fungus, or a saprophyte yeast of the digestive tract; in 1996, it is placed in the group of stramenopiles (heterokonts). Since its new classification, many questions have arisen around this protist about its role as a pathogen or non-pathogen organism. Recent evidence indicates that Blastocystis spp. participates in the immune inflammatory response in the intestinal microbiome generating an anti-inflammatory response, showing a lower concentration of fecal inflammatory markers in infected human hosts. Here, we review recent findings on the regulatory function of Blastocystis spp. in the immune inflammatory response to comprehend the purpose of Blastocystis spp. in health and disease, defining if Blastocystis spp. is really a pathogen, a commensal or even a mutualist in the human gut microbiome.

High Prevalence and Diversity of Caliciviruses in a Community Setting Determined by a Metagenomic Approach.

We recently carried out a metagenomic study to determine the fecal virome of infants during their first year of life in a semirural community in Mexico. A total of 97 stool samples from nine children were collected starting 2 weeks after birth and monthly thereafter until 12 months of age. In this work, we describe the prevalence and incidence of caliciviruses in this birth cohort. We found that 54 (56%) and 24 (25%) of the samples were positive for norovirus and sapovirus sequence reads detected by next-generation sequencing, respectively. Potential infections were arbitrarily considered when at least 20% of the complete virus genome was determined. Considering only these samples, there were 3 cases per child/year for norovirus and 0.33 cases per child/year for sapovirus. All nine children had sequence reads related to norovirus in at least 2 and up to 10 samples, and 8 children excreted sapovirus sequence reads in 1 and up to 5 samples during the study. The virus in 35 samples could be genotyped. The results showed a high diversity of both norovirus (GI.3[P13], GI.5, GII.4, GII.4[P16], GII.7[P7], and GII.17[P17]) and sapovirus (GI.1, GI.7, and GII.4) in the community. Of interest, despite the frequent detection of caliciviruses in the stools, all children remained asymptomatic during the study. Our results clearly show that metagenomic studies in stools may reveal a detailed picture of the prevalence and diversity of gastrointestinal viruses in the human gut during the first year of life. IMPORTANCE Human caliciviruses are important etiological agents of acute gastroenteritis in children under 5 years of age. Several studies have characterized their association with childhood diarrhea and their presence in nondiarrheal stool samples. In this work, we used a next-generation sequencing approach to determine, in a longitudinal study, the fecal virome of infants during their first year of life. Using this method, we found that caliciviruses can be detected significantly more frequently than previously reported, providing a more detailed picture of the prevalence and genetic diversity of these viruses in the human gut during early life.

Exposure to Parasitic Protists and Helminths Changes the Intestinal Community Structure of Bacterial Communities in a Cohort of Mother-Child Binomials from a Semirural Setting in Mexico.

An estimated 3.5 billion people are colonized by intestinal parasites worldwide. Intestinal parasitic eukaryotes interact not only with the host but also with the intestinal microbiota. In this work, we studied the relationship between the presence of multiple enteric parasites and the community structures of gut bacteria and eukaryotes in an asymptomatic mother-child cohort from a semirural community in Mexico. Fecal samples were collected from 46 mothers and their respective children, with ages ranging from 2 to 20 months. Mothers and infants were found to be multiparasitized by Blastocystis hominis, Entamoeba dispar, Endolimax nana, Chilomastix mesnili, Iodamoeba butshlii, Entamoeba coli, Hymenolepis nana, and Ascaris lumbricoides. Sequencing of bacterial 16S rRNA and eukaryotic 18S rRNA genes showed a significant effect of parasite exposure on bacterial beta-diversity, which explained between 5.2% and 15.0% of the variation of the bacterial community structure in the cohort. Additionally, exposure to parasites was associated with significant changes in the relative abundances of multiple bacterial taxa, characterized by an increase in Clostridiales and decreases in Actinobacteria and Bacteroidales. Parasite exposure was not associated with changes in intestinal eukaryote relative abundances. However, we found several significant positive correlations between intestinal bacteria and eukaryotes, including Oscillospira with Entamoeba coli and Prevotella stercorea with Entamoeba hartmanni, as well as the co-occurrence of the fungus Candida with Bacteroides and Actinomyces, Bifidobacterium, and Prevotella copri and the fungus Pichia with Oscillospira. The parasitic exposure-associated changes in the bacterial community structure suggest effects on microbial metabolic routes, host nutrient uptake abilities, and intestinal immunity regulation in host-parasite interactions. IMPORTANCE The impact of intestinal eukaryotes on the prokaryotic microbiome composition of asymptomatic carriers has not been extensively explored, especially in infants and mothers with multiple parasitic infections. In this work, we studied the relationship between protist and helminth parasite colonization and the intestinal microbiota structure in an asymptomatic population of mother-child binomials from a semirural community in Mexico. We found that the presence of parasitic eukaryotes correlated with changes in the bacterial gut community structure in the intestinal microbiota in an age-dependent way. Parasitic infection was associated with an increase in the relative abundance of the class Clostridia and decreases of Actinobacteria and Bacteroidia. Parasitic infection was not associated with changes in the eukaryote community structure. However, we observed strong positive correlations between bacterial and other eukaryote taxa, identifying novel relationships between prokaryotes and fungi reflecting interkingdom interactions within the human intestine.

The gut virome of healthy children during the first year of life is diverse and dynamic.

In this work, we determined the diversity and dynamics of the gut virome of infants during the first year of life. Fecal samples were collected monthly, from birth to one year of age, from three healthy children living in a semi-rural village in Mexico. Most of the viral reads were classified into six families of bacteriophages including five dsDNA virus families of the order Caudovirales, with Siphoviridae and Podoviridae being the most abundant. Eukaryotic viruses were detected as early as two weeks after birth and remained present all along the first year of life. Thirty-four different eukaryotic virus families were found, where eight of these families accounted for 98% of all eukaryotic viral reads: Anelloviridae, Astroviridae, Caliciviridae, Genomoviridae, Parvoviridae, Picornaviridae, Reoviridae and the plant-infecting viruses of the Virgaviridae family. Some viruses in these families are known human pathogens, and it is surprising that they were found during the first year of life in infants without gastrointestinal symptoms. The eukaryotic virus species richness found in this work was higher than that observed in previous studies; on average between 7 and 24 virus species were identified per sample. The richness and abundance of the eukaryotic virome significantly increased during the second semester of life, probably because of an increased environmental exposure of infants with age. Our findings suggest an early and permanent contact of infants with a diverse array of bacteriophages and eukaryotic viruses, whose composition changes over time. The bacteriophages and eukaryotic viruses found in these children could represent a metastable virome, whose potential influence on the development of the infant's immune system or on the health of the infants later in life, remains to be investigated.

Differential Pathogenic Gene Expression of E. histolytica in Patients with Different Clinical Forms of Amoebiasis.

The etiological agent of human amoebiasis is the protozoan parasite E. histolytica; the disease is still an endemic infection in some countries and the outcome of infection in the host infection can range from asymptomatic intestinal infection to intestinal or liver invasive forms of the disease. The invasive character of this parasite is multifactorial and mainly due to the differential expression of multiple pathogenic genes. The aim of the present work was to measure the differential expression of some genes in different specimens of patients with amoebic liver abscess (ALA) and specimens of genital amoebiasis (AG) by RT-qPCR. Results show that the expression of genes is different in both types of samples. Almost all studied genes were over expressed in both sets of patients; however, superoxide dismutase (Ehsod), serine threonine isoleucine rich protein (Ehstirp), peroxiredoxin (Ehprd) and heat shock protein 70 and 90 (Ehhsp-70, EHhsp-90) were higher in AG biopsies tissue. Furthermore, cysteine proteinases 5 and 2 (Ehcp5, Ehcp2), lectin (Ehgal/galnaclectin) and calreticulin (Ehcrt) genes directly associate with pathogenic mechanisms of E. histolytica had similar over expression in both AG and ALA samples. In summary the results obtained show that trophozoites can regulate the expression of their genes depending on stimuli or environmental conditions, in order to regulate their pathogenicity and ensure their survival in the host.

Tobamoviruses can be frequently present in the oropharynx and gut of infants during their first year of life.

Plant viruses have been reported to be common in the gut of human adults, presumably as result of food ingestion. In this work, we report that plant viruses can also be found frequently in the gut and oropharynx of children during their first year of life, even when they are exclusively breast-fed. Fecal and oropharynx samples were collected monthly, from birth to 1 year of age, from three apparently healthy children in a semi-rural community and analyzed by next generation sequencing. In 100% of the fecal samples and 65% of the oropharynx samples at least one plant virus was identified. Tobamoviruses in the Virgaviridae family were by far the most frequently detected, with tropical soda apple mosaic virus, pepper mild mottle virus, and opuntia tobamovirus 2 being the most common species. Seventeen complete virus genomes could be assembled, and phylogenetic analyses showed a large diversity of virus strains circulating in the population. These results suggest that children are continuously exposed to an extensive and highly diverse collection of tobamoviruses. Whether the common presence of plant viruses at an early age influences the infant's immune system, either directly or through interaction with other members of the microbiota, remains to be investigated.

Use of next-generation amplicon sequencing to study Blastocystis genetic diversity in a rural human population from Mexico.

BACKGROUND: The intestinal parasite Blastocystis is found in humans and animals around the world. It is spread through the consumption of contaminated food and water and has been associated with a variety of intestinal symptoms. Blastocystis is one of the most common intestinal parasites in humans, yet its prevalence and distribution in humans in North America is not well characterized. METHODS: Next-generation amplicon sequencing of a region of the Blastocystis SSU rRNA gene was applied to DNA extracted from fecal specimens obtained from 182 inhabitants of a rural population in Mexico to characterize Blastocystis prevalence, subtype distribution, and intra-host subtype diversity in humans. RESULTS: Of the 182 samples tested in this study, 68.1% (124) contained one or more Blastocystis subtypes. Subtype 3 was the most common subtype observed and was found in 81.5% of the positive samples. Subtype 1, 16.9% of the positive samples, and subtype 2, 17.7% of the positive samples, were also found in this population. Mixed infections were observed in 13.7% of the positive samples. In this population, the odds of having Blastocystis increased in adulthood (> 15 years; OR: 1.72, P < 0.0001), and the odds of having subtype 1 increased in the presence of farm animals (OR: 1.51, P = 0.03). The odds of having subtype 1, subtype 2, or a mixed infection decreased in the presence of cement flooring (OR: - 1.61, P = 0.005; OR: - 1.14, P = 0.03; OR: - 1.48, P = 0.02) possibly indicating socioeconomic factors are involved in the risk of acquiring one of these subtypes. CONCLUSIONS: These data contribute to our understanding of the epidemiology of Blastocystis infection in humans and can be used to shape future studies which aim to better characterize the transmission pathways and health outcomes of Blastocystis infections.

Correction: Differential expression of pathogenic genes of Entamoeba histolytica vs E. dispar in a model of infection using human liver tissue explants.

[This corrects the article DOI: 10.1371/journal.pone.0181962.].

Entamoeba histolytica Calreticulin Induces the Expression of Cytokines in Peripheral Blood Mononuclear Cells Isolated From Patients With Amebic Liver Abscess.

Calreticulin (CRT) is a highly conserved protein in the endoplasmic reticulum that plays important roles in the regulation of key cellular functions. Little is known about the participation of E. histolytica CRT (EhCRT) in the processes of pathogenicity or in the modulation of the host immune response. The aim of this study was to evaluate the role of CRT in the proliferation and the cytokine profile in peripheral blood mononuclear cells (PBMCs) from patients with amebic liver abscess (ALA) during the acute phase (AP-ALA) of the disease compared to patients during the resolution phase (R-ALA). The PBMCs from each participant were cocultured with EhCRT and tested by the colorimetric method to evaluate their proliferation index (PI). The supernatants were subjected to an enzyme-linked immunosorbent assay (ELISA) to evaluate the concentration of cytokines. The mean values of all groups were compared using the independent t-test. When the PIs of individuals without diagnosis of liver abscess (NEG) were compared, there were no statistically significant differences in the proliferation of PBMCs between patients with AP-ALA and R-ALA when stimulated with EhCRT or concanavalin A (ConA). However, the levels of interleukins [IL-6, IL-10, granulocyte colony stimulating factor (GCSF), and transforming growth factor ß1 (TGFß1)] were higher in patients with AP-ALA, whereas in patients with R-ALA, higher levels of interferon gamma (IFNγ) were detected. These results suggest that EhCRT acts as a mitogen very similar to the activity of ConA. In addition, EhCRT is an excellent immunogen for the specific activation of PBMCs, inducing the differential expression of ILs depending on the outcome of disease, determining the type of immune response: a Th2 cytokine profile during the acute phase and a Th1 profile during the resolution phase.

Genetic Diversity and Distribution of Blastocystis Subtype 3 in Human Populations, with Special Reference to a Rural Population in Central Mexico.

Blastocystis subtype 3 (ST3) is a parasitic protist found in the digestive tract of symptomatic and asymptomatic humans around the world. While this parasite exhibits a high prevalence in the human population, its true geographic distribution and global genetic diversity are still unknown. This gap in knowledge limits the understanding of the spread mechanisms, epidemiology, and impact that this parasite has on human populations. Herein, we provided new data on the geographical distribution and genetic diversity of Blastocystis ST3 from a rural human population in Mexico. To do so, we collected and targeted the SSU-rDNA region in fecal samples from this population and further compared its genetic diversity and structure with that previously observed in populations of Blastocystis ST3 from other regions of the planet. Our analyses reveled that diversity of Blastocystis ST3 showed a high haplotype diversity and genetic structure to the world level; however, they were low in the Morelos population. The haplotype network revealed a common widespread haplotype from which the others were generated recently. Finally, our results suggested a recent expansion of the diversity of Blastocystis ST3 worldwide.

Differential expression of pathogenic genes of Entamoeba histolytica vs E. dispar in a model of infection using human liver tissue explants.

We sought to establish an ex vivo model for examining the interaction of E. histolytica with human tissue, using precision-cut liver slices (PCLS) from donated organs. E. histolytica- or E. dispar-infected PCLS were analyzed at different post-infection times (0, 1, 3, 24 and 48 h) to evaluate the relation between tissue damage and the expression of genes associated with three factors: a) parasite survival (peroxiredoxin, superoxide dismutase and 70 kDa heat shock protein), b) parasite virulence (EhGal/GalNAc lectin, amoebapore, cysteine proteases and calreticulin), and c) the host inflammatory response (various cytokines). Unlike E. dispar (non-pathogenic), E. histolytica produced some damage to the structure of hepatic parenchyma. Overall, greater expression of virulence genes existed in E. histolytica-infected versus E. dispar-infected tissue. Accordingly, there was an increased expression of EhGal/GalNAc lectin, Ehap-a and Ehcp-5, Ehcp-2, ehcp-1 genes with E. histolytica, and a decreased or lack of expression of Ehcp-2, and Ehap-a genes with E. dispar. E. histolytica-infected tissue also exhibited an elevated expression of genes linked to survival, principally peroxiredoxin, superoxide dismutase and Ehhsp-70. Moreover, E. histolytica-infected tissue showed an overexpression of some genes encoding for pro-inflammatory interleukins (ILs), such as il-8, ifn-γ and tnf-α. Contrarily, E. dispar-infected tissue displayed higher levels of il-10, the gene for the corresponding anti-inflammatory cytokine. Additionally, other genes were investigated that are important in the host-parasite relationship, including those encoding for the 20 kDa heat shock protein (HSP-20), the AIG-1 protein, and immune dominant variable surface antigen, as well as for proteins apparently involved in mechanisms for the protection of the trophozoites in different environments (e.g., thioredoxin-reductase, oxido-reductase, and 9 hypothetical proteins). Some of the hypothetical proteins evidenced interesting overexpression rates, however we should wait to their characterization. This finding suggest that the present model could be advantageous for exploring the complex interaction between trophozoites and hepatocytes during the development of ALA, particularly in the initial stages of infection.