Identification and typing of free-living Acanthamoeba spp. by MALDI-TOF MS Biotyper.

Over the years, the potential pathogenicity of Acanthamoeba for humans and animals has gained increasing attention from the scientific community. More than 24 species belong to this genus, however only some of them are causative agents of keratitis and encephalitis in humans. Due to technical difficulties in diagnosis, these infections are likely to be under-detected. The introduction of 18S rDNA amplification for the identification of Acanthamoeba has dramatically enhanced diagnosis performances, but the attestation of genotyping requires supplementary sequencing-based procedures. In this study, 15 Acanthamoeba strains were collected and grown on nutrient agar media. Each strain was genotyped by end-point PCR assay for the amplification of the 18S rDNA gene and the genotype was assigned by sequencing analysis through neighbor joining phylogenetic tree. In order to optimize standardization of the MALDI-TOF MS assay, we established the collection time point at the cystic phase. Two strains of each genotype were randomly chosen to customize the biotyper database. For all strains, 24 spectral measurements were acquired and submitted to identification and cluster analysis of spectra. The obtained results highlighted the correct identification of Acanthamoeba strains and the overlapping of spectra dendrogram clusters to the 18S genotype assignations. In conclusion, the MALDI-TOF MS Biotyper revealed the capability to identify and genotype the Acanthamoeba strains, providing a new frontier in the diagnostic identification of amaebae and in taxonomic and phylogenetic studies.

Genotypic heterogeneity based on 18S-rRNA gene sequences among Acanthamoeba isolates from clinical samples in Italy.

Acanthamoeba keratitis (AK) is an ocular disease caused by members of a genus of free-living amoebae and it is associated predominantly with contact lens (CL) use. This study reports 55 cases of AK diagnosed in Italy. Genotype identification was carried out by PCR assay followed by sequence analysis of the 18S rRNA gene using the genus specific primers JDP1 and JDP2. Genotype assignment was based on phenetic analysis of the ASA.S1 subset of the small-subunit rRNA gene sequences. The material has been collected at the Polyclinic Tor Vergata of Rome for a total of 19 isolates and at the Polyclinic Hospital of Bari (36 isolates). Thirty-three out of the 55 genetically characterized isolates were assigned to the genotype T4. Ten isolates were identified as belonging to the genotype T15 thus confirming the first association between the genotype T15 and human amoebic keratitis previously described from the same area. We underline the occurrence of the genotype T3 and T11 identified for the first time in the country.

A One-Year Retrospective Analysis of Viral and Parasitological Agents in Wildlife Animals Admitted to a First Aid Hospital.

This study aimed to provide information on the presence and frequency of viral and parasitic agents in wildlife presented to a Veterinary Teaching Hospital in 2020-2021. Serum and faecal samples were collected from 50 rescued animals (roe deer, fallow deer, foxes, badgers, pine martens, and porcupines) and examined by serological, molecular, and parasitological techniques. Transtracheal wash (TTW) was also collected post-mortem from roe deer. Overall, the results of the different techniques showed infections with the following viral and parasitic agents: Bovine Viral Diarrhea Virus, Small Ruminant Lentiviruses, Kobuvirus, Astrovirus, Canine Adenovirus 1, Bopivirus, gastrointestinal strongyles, Capillaria, Ancylostomatidae, Toxocara canis, Trichuris vulpis, Hymenolepis, Strongyloides, Eimeria, Isospora, Dictyocaulus, Angiostrongylus vasorum, Crenosoma, Dirofilaria immitis, Neospora caninum, Giardia duodenalis, and Cryptosporidium. Sequencing (Tpi locus) identified G. duodenalis sub-assemblages AI and BIV in one roe deer and one porcupine, respectively. Adult lungworms collected from the TTW were identified as Dictyocaulus capreolus (COX1 gene). This is the first molecular identification of G. duodenalis sub-assemblage AI and D. capreolus in roe deer in Italy. These results show a wide presence of pathogens in wild populations and provide an overview of environmental health surveillance.

Investigating the infant gut microbiota in developing countries: worldwide metagenomic meta-analysis involving infants living in sub-urban areas of Côte d'Ivoire.

In recent decades, infants' gut microbiota has aroused constant scientific interest, primarily due to early- and long-term repercussions on the host health. In this context, nutritional challenges such as those found in less developed countries can influence infants' gut microbiota development, thus generating potentially critical health outcomes. However, comprehensive investigations regarding species-level differences in the infant gut microbiota's composition between urbanized and rural countries are still missing. In this study, 16S rRNA and Shallow Shotgun metagenomics sequencing were exploited to dissect the microbial community's species-level composition of 11 faecal samples collected from infants living in a semi-urban area of Sub-Saharan Africa, i.e. Côte d'Ivoire. Moreover, the generated data were coupled with those retrieved from public available metagenomic repositories, including two rural communities and 13 urban communities of industrialized countries. The meta-analysis led to the identification of Infant Species Community States Type (ISCSTs) and microbial species covariances, which were exploited to reveal key signatures of infants living in rural and semi-urban societies. Remarkably, analysis of rural and semi-urban datasets revealed shifts from ISCSTs prevalent in urbanized populations with putative health implications. Thus, indicating the need for population-wide investigations aimed to define the factors determining such potentially harmful gut microbial communities' signatures.

Analysis of Human Gut Microbiota Composition Associated to the Presence of Commensal and Pathogen Microorganisms in Côte d'Ivoire.

BACKGROUND: The human gut microbiota is a microbial ecosystem contributing to the maintenance of host health with functions related to immune and metabolic aspects. Relations between microbiota and enteric pathogens in sub-Saharan Africa are scarcely investigated. The present study explored gut microbiota composition associated to the presence of common enteric pathogens and commensal microorganisms, e.g., Blastocystis and Entamoeba species, in children and adults from semi-urban and non-urban localities in Côte d'Ivoire. METHODS: Seventy-six stool samples were analyzed for microbiota composition by 16S rRDNA sequencing. The presence of adeno-, entero-, parechoviruses, bacterial and protozoal pathogens, Blastocystis, and commensal Entamoeba species, was analyzed by different molecular assays. RESULTS: Twelve individuals resulted negative for any tested microorganisms, 64 subjects were positive for one or more microorganisms. Adenovirus, enterovirus, enterotoxigenic Escherichia coli (ETEC), and Blastocystis were frequently detected. CONCLUSIONS: The bacterial composition driven by Prevotellaceae and Ruminococcaceae confirmed the biotype related to the traditional dietary and cooking practices in low-income countries. Clear separation in UniFrac distance in subjects co-harboring Entamoeba hartmanni and Blastocystis was evidenced. Alpha diversity variation in negative control group versus only Blastocystis positive suggested its possible regulatory contribution on intestinal microbiota. Pathogenic bacteria and virus did not affect the positive outcome of co-harbored Blastocystis.

Correction: Lower prevalence of Blastocystis sp. infections in HIV positive compared to HIV negative adults in Ghana.

[This corrects the article DOI: 10.1371/journal.pone.0221968.].

Lower prevalence of Blastocystis sp. infections in HIV positive compared to HIV negative adults in Ghana.

BACKGROUND: Sub-Saharan Africa is endemic for intestinal parasites and distinguished for the largest burden of HIV cases. Blastocystis sp. is one of the most common protists infecting humans but its role in human disease is still controversial. Aim of this study was to investigate the prevalence of Blastocystis sp. in HIV positive and negative adults in Ghana and its association with immune status and other risk factors. METHODS: 122 HIV positive outpatients and 70 HIV negative blood donors from the Komfo Anokye Teaching Hospital in Kumasi, Ghana, were included in the present study. Demographic, clinical and laboratory data were collected and HIV positive patients distinguished for CD4+ T cell count <200 cells/µl (n = 54) and >200 cells/µl (n = 68). A Blastocystis's phylogenetic analysis was performed to determine sample subtype (ST). RESULTS: The prevalence of Blastocystis sp. in adult HIV positive individuals was lower than in HIV negative persons (6.6% vs. 20.0%, p = 0.008) and Blastocystis sp. ST1 was the most prevalent strain. Within HIV positive participants, the prevalence of Blastocystis sp. was lower in those individuals with CD4+ T cell count <200 cells/µl than in patients with higher CD4+ T cell count (1.9% vs. 10.3%, p = 0.076). Multiple regression analysis revealed that Blastocystis sp. was inversely associated with an obese Body Mass Index (BMI) in HIV negative persons (p = 0.040). Presence of Blastocystis sp. was correlated with higher CD4+ T cell count in HIV positive participants (p = 0.049). CONCLUSION: It is largely reported that people living with HIV (PLHIV) in Africa are affected from parasite infections and that co-infections may adversely impact on their immune status, accelerating progress to AIDS and worsening gastrointestinal manifestations. Differently, in this study Blastocystis sp. was associated with a better immune status jointly with a healthy body weight while it seems to be reduced with the progression of HIV infection. This data agree with recent suggestions that Blastocystis sp. can represent a component of the healthy gut microbiota.

Genome Sequence of Novel Human Parechovirus Type 17.

Human parechoviruses (HPeV) circulate worldwide, causing a broad variety of symptoms, preferentially in early childhood. We report here the nearly complete genome sequence of a novel HPeV type, consisting of 7,062 nucleotides and encoding 2,179 amino acids. M36/CI/2014 was taxonomically classified as HPeV-17 by the picornavirus study group.

Gut microbiota related to Giardia duodenalis, Entamoeba spp. and Blastocystis hominis infections in humans from Côte d'Ivoire.

INTRODUCTION: Literature data provide little information about protozoa infections and gut microbiota compositional shifts in humans. This preliminary study aimed to describe the fecal bacterial community composition of people from Côte d'Ivoire harboring Giardia duodenalis, Entamoeba spp., and Blastocystis hominis, in trying to discover possible alterations in their fecal microbiota structure related to the presence of such parasites. METHODOLOGY: Twenty fecal samples were collected from people inhabiting three different localities of Côte d'Ivoire for copromicroscopic analysis and molecular identification of G. duodenalis, Entamoeba spp., and B. hominis. Temporal temperature gradient gel electrophoresis (TTGE) was used to obtain a fingerprint of the overall bacterial community; quantitative polymerase chain reaction (qPCR) was used to define the relative abundances of selected bacterial species/group, and multivariate statistical analyses were employed to correlate all data. RESULTS: Cluster analysis revealed a significant separation of TTGE profiles into four clusters (p < 0.0001), with a marked difference for G. duodenalis-positive samples in relation to the others (p = 5.4×10-6). Interestingly, qPCR data showed how G. duodenalis-positive samples were related to a dysbiotic condition that favors potentially harmful species (such as Escherichia coli), while Entamoeba spp./B. hominis-positive subjects were linked to a eubiotic condition, as shown by a significantly higher Faecalibacterium prausnitzii-Escherichia coli ratio. CONCLUSIONS: This preliminary investigation demonstrates a differential fecal microbiota structure in subjects infected with G. duodenalis or Entamoeba spp./B. hominis, paving the way for using further next-generation DNA technologies to better understand host-parasite-bacteria interactions, aimed at identifying potential indicators of microbiota changes.

The Gly972-->Arg IRS-1 variant is associated with type 1 diabetes in continental Italy.

The Arg(972) insulin receptor substrate-1 (IRS-1) variant has been hypothesized to play a role in pancreatic beta-cell stimulus-coupled insulin secretion and survival. We analyzed the relations between type 1 diabetes and the Arg(972) IRS-1 variant. The frequency of the IRS-1 Arg(972) variant was investigated in two independent sets of unrelated patients: a case-control study and a collection of type 1 diabetes simplex families. In the former group, frequency of the IRS-1 Arg(972) variant was significantly increased in the patients (P = 0.0008), conferring an OR of 2.5. Transmission disequilibrium analysis of data obtained from the family set revealed that the Arg(972) IRS-1 variant was transmitted from heterozygous parents to affected probands at a frequency of 70.2% (P < 0.02). Arg(972) IRS-1 frequency showed no significant correlation with HLA genotypic risk for type 1 diabetes. Arg(972) IRS-1 type 1 diabetic patients also had lower fasting plasma concentrations of C-peptide at the time of diagnosis with respect to patients carrying the wild-type IRS-1 (0.49 +/- 0.058, n = 34, and 0.76 +/- 0.066, n = 134, respectively [means +/- SE]; P = 0.051). Our findings suggest a role for Arg(972) IRS-1 in conferring risk for the development of type 1 diabetes.

Application of Luminex Gastrointestinal Pathogen Panel to human stool samples from Côte d'Ivoire.

INTRODUCTION: Gastrointestinal infections caused by viruses, bacteria, and parasites are endemic in most developing countries due to inadequate provision of safe water supplies, sanitation, and hygiene. To investigate the enteric pathogens infecting people living in Côte d'Ivoire, the Luminex Gastrointestinal Pathogen Panel (xTAG GPP) assay was used to analyze 34 human fecal samples. This study represents the first application of this technology to samples from a sub-Saharan African country. METHODOLOGY: Thirty-four stool samples from asymptomatic and symptomatic patients, 1-15 years of age, were analyzed by xTAG GPP. The Luminex assay represents a qualitative bead-based multiplexed molecular diagnostic test able to identify concurrently 15 enteric pathogens, including bacteria, viruses, and parasites. RESULTS: Overall, 22 out of 34 (64.7%) fecal specimens were detected to be positive by xTAG GPP. Sixteen were from asymptomatic subjects, and 10 patients (45.4%) showed co-infections. G. duodenalis was detected in 15 patients, in both mono- and co-infections, representing the most frequent pathogen, followed by enterotoxigenic Escherichia coli (ETEC) LT/ST. Four norovirus isolates were also detected and assigned to genogroups I and II. CONCLUSIONS: Considering the burden of enteric infections in developing countries, particularly among children, and the high rate of co-infections in asymptomatic subjects, this study shows the need for diagnostic tools such as xTAG GPP to improve diagnosis and treatment of these infections in endemic areas.

Isolation and molecular characterization of free-living amoebae from different water sources in Italy.

Free-living amoebae (FLA) are protozoa ubiquitous in Nature, isolated from a variety of environments worldwide. In addition to their natural distribution, some species have been found to be pathogenic to humans. In the present study a survey was conducted in order to evaluate the presence and to characterize at molecular level the isolates of amoebic organisms collected from different water sources in Italy. A total of 160 water samples were analyzed by culture and microscopic examination. FLA were found in 46 (28.7%) of the investigated water samples. Groundwater, well waters, and ornamental fountain waters were the sources with higher prevalence rates (85.7%, 50.0%, and 45.9%, respectively). Identification of FLA species/genotypes, based on the 18S rDNA regions, allowed to identify 18 (39.1%) Acanthamoeba isolates (genotypes T4 and T15) and 21 (45.6%) Vermamoeba vermiformis isolates. Other FLA species, including Vahlkampfia sp. and Naegleria spp., previously reported in Italy, were not recovered. The occurrence of potentially pathogenic free-living amoebae in habitats related to human population, as reported in the present study, supports the relevance of FLA as a potential health threat to humans.

Detection and characterization of enteroviruses and parechoviruses in healthy people living in the South of Côte d'Ivoire.

BACKGROUND: Human enteroviruses (EVs) and parechoviruses (HPeVs) belong to the family Picornaviridae. Although most EV and HPeV infections remain asymptomatic, both pathogens can cause a wide spectrum of clinical manifestations ranging from respiratory or gastrointestinal symptoms to myocarditis, neonatal sepsis, and infections of the central nervous system. OBJECTIVES: Aim of the present study was to investigate the spectrum of EVs and HPeVs in apparently healthy adults and children living in the South of Côte d'Ivoire. STUDY DESIGN: The study included 105 stool samples obtained from healthy individuals aged 0-53 years between June 2013 and December 2014 in the Sud-Como region of Côte d'Ivoire. After collection and shipment to Germany, the samples were analyzed by real-time PCR for the presence of EVs and HPeVs RNA. Molecular typing and virus isolation of all samples were performed.''é RESULTS: Out of 105 samples, 24 (22.8%) were EV positive and six (5.2%) were HPeV positive. Twenty-one EV positive samples could be characterized with serotypes belonging to EV group A-C, while three could not be further specified. Interestingly, several rarely described serotypes were identified, e.g., EV-C99, EV-B93, EV-C116, and EV-A119. Typing of HPeV positive samples resulted in HPeV-1 and -5 detections, while one isolate could not be assigned to the known HPeV types. CONCLUSIONS: This study showed a large variety of EV strains in healthy people in the South of Côte d'Ivoire and provided the first available data about HPeV infections in a sub-Saharan African country.

The Arg972 variant in insulin receptor substrate-1 is associated with an atherogenic profile in offspring of type 2 diabetic patients.

The insulin receptor substrate-1 (IRS-1) gene has been considered a candidate for insulin resistance, type 2 diabetes, and coronary artery disease. To investigate the relationship between the common Gly(972)Arg IRS-1 variant and the presence of cardiovascular risk factors, 153 glucose-tolerant, unrelated offspring of type 2 diabetic patients were studied. There were no differences between Arg(972) IRS-1 carriers and noncarriers in age, gender, body mass index, waist/hip ratio, body composition, fasting glucose and insulin levels, and glucose or insulin levels during the oral glucose tolerance test. Insulin sensitivity, assessed by hyperinsulinemic-euglycemic clamp, was significantly reduced in carriers of Arg(972) IRS-1 (P < 0.03). Carriers of Arg(972) IRS-1 displayed many features of the insulin resistance syndrome, including higher values for serum triglycerides (P < 0.01), total/high density lipoprotein cholesterol ratio (P < 0.01), free fatty acid levels (P < 0.04), systolic blood pressure (P < 0.04), microalbuminuria (P < 0.003), and intima-media thickness (P < 0.02). These results suggest that the Arg(972) IRS-1 variant could contribute to the risk for atherosclerotic cardiovascular diseases associated with type 2 diabetes by producing a cluster of insulin resistance-related metabolic abnormalities.

Polymorphisms of the insulin receptor substrate-2 in patients with type 2 diabetes.

We investigated the significance of Gly1057Asp and Leu647Val insulin receptor substrate (IRS)-2 polymorphisms in two Italian cohorts comprising 186 glucose-tolerant subjects and 240 subjects with type 2 diabetes from the Lazio region (i.e. representative of central Italy), and 123 glucose-tolerant subjects from the Sicily region (i.e. representative of south Italy). The allelic frequency of Gly1057Asp variant did not differ between diabetics (32.9%) and nondiabetic subjects, whatever their ethnicity was (35.8% and 33.7% from Lazio and Sicily, respectively). As compared with Gly/Gly subjects within each group, Asp/Asp individuals showed no differences in quantitative traits, including fasting insulin and C-peptide, and several indices of insulin sensitivity and secretion. Only one of the diabetic patients was heterozygous for the Leu647Val variant, and none of the control subjects carried this variant. This patient had three children who were also heterozygous for this variant. They were glucose tolerant, and their insulin sensitivity and insulin secretion indices were within the range of age-matched controls. We also analyzed IRS-2 function in fibroblasts from carriers of Gly1057Asp or Leu647Val variant. No defects in IRS-2 expression, insulin-stimulated phosphorylation, or binding to the p85 subunit of phosphatidylinositol 3-kinase were observed. These results strongly argue against a major role of IRS-2 polymorphisms in the pathogenesis of type 2 diabetes.

Giardia duodenalis genotypes and Cryptosporidium species in humans and domestic animals in Côte d'Ivoire: occurrence and evidence for environmental contamination.

Giardia duodenalis genotypes and Cryptosporidium species were studied in humans and free-ranging animals living in closed enclaves in Côte d'Ivoire. Three hundred and seven stool samples were tested from humans, and 47 from freely roaming domestic animals (dogs, goats, ducks, chickens). Molecular characterization of the isolates was performed by sequence analysis of a portion of the SSU-rDNA for Giardia and the COWP gene for Cryptosporidium, and a ß-giardin SYBR-green real-time PCR was also used to confirm the assignment of Giardia isolates to Assemblages. In humans, genotyping of Giardia assigned many of the sequences (43/56 by the SSU-rDNA gene, and 36/61 by the ß-giardin gene) to Assemblage B. The animal species harboured only zoonotic Assemblages A and B, except for dogs, in which host specific Assemblages C and D were also detected. Cryptosporidium meleagridis, C. parvum and C. hominis were detected in humans, while among the animals only chickens were found positive for oocysts, identified as C. meleagridis and C. parvum. The results provide further evidence about the role of free-ranging domestic animals living closely with humans in the environmental dissemination and potential transmission of these anthropozoonotic pathogens to humans.

Decreased IRS2 and TIMP3 expression in monocytes from offspring of type 2 diabetic patients is correlated with insulin resistance and increased intima-media thickness.

OBJECTIVE: In humans, it is unclear if insulin resistance at the monocyte level is associated with atherosclerosis in vivo. Here we have studied first-degree relatives of patients with type 2 diabetes to investigate whether a reduction in components of the insulin signal transduction pathways, such as the insulin receptor (InsR) or InsR substrate 1 or 2 (IRS1 or IRS2), or a reduction in genetic modifiers of insulin action, such as the TIMP3/ADAM17 (tissue inhibitor of metalloproteinase 3/A disintegrin and metalloprotease domain 17) pathway, is associated with evidence of atherosclerosis. RESEARCH DESIGN AND METHODS: Insulin sensitivity was analyzed through euglycemic-hyperinsulinemic clamp, and subclinical atherosclerosis was analyzed through intimal medial thickness. Monocytes were isolated through magnetic cell sorting, and mRNA and proteins were extracted and analyzed by quantitative PCR and pathscan enzyme-linked immunosorbent assays, respectively. RESULTS: In monocyte cells from human subjects with increased risk for diabetes and atherosclerosis, we found that gene expression, protein levels, and tyrosine phosphorylation of IRS2, but not InsR or IRS1, were decreased. TIMP3 was also reduced, along with insulin resistance, resulting in increased ectodomain shedding activity of the metalloprotease ADAM17. CONCLUSIONS: Systemic insulin resistance and subclinical atherosclerosis are associated with decreased IRS2 and TIMP3 expression in circulating monocytes.