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.

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.

Giardia duodenalis in Alpine (Rupicapra rupicapra rupicapra) and Apennine (Rupicapra pyrenaica ornata) chamois.

BACKGROUND: Although chamois Rupicapra spp. are the most abundant mountain ungulates in Europe, no data are available on the presence of Giardia duodenalis infecting these species. METHODS: A total of 157 fecal samples from Alpine Rupicapra rupicapra rupicapra and Apennine Rupicapra pyrenaica ornata chamois were tested for the presence of G. duodenalis by immunofluorescence test, quantitative Real Time PCR and end-point PCR for genotype characterization. RESULTS: G. duodenalis was detected in R. r. rupicapra and R. p. ornata, with a percentage value of 4.45 (5.82 and 1.85 %, respectively), and a cyst burden of up to 31,800 cysts/g of feces. Assemblages A/AI and E were identified in R. r. rupicapra and assemblage A/AIII in R. p. ornata. CONCLUSIONS: The present study represents the first record of Giardia duodenalis in Rupicapra spp., suggesting that these wild bovids can play an epidemiological role in environmental contamination and transmission of both zoonotic and non-zoonotic genotypes.

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.

A waterborn zoonotic helminthiase in an Italian diver: a case report of a cutaneous Sparganum infection and a review of European cases.

Many waterborne helminthes are opportunistic parasites that can travel directly from animals to man and may contain forms capable of penetrating the skin. Among these, Sparganum is the pseudophyllidean tapeworm that belongs to the genus Spirometra, which is responsible for parasitic zoonosis; it is rarely detected in Europe and is caused by the plerocercoid infective larva. Thus far, only six cases of cutaneous and ocular sparganosis have been reported in Europe; two and four cases have occurred in France and Italy, respectively. Herein, we describe a new case of sparganosis in Italy that affected a male diver who presented to the Bambino Gesù Children's Hospital of Rome. The patient's skin biopsy was submitted to the Parasitology department who, in consultation with Pathology, concluded that the morphologic and microscopic findings were those of Sparganum spp. larvae. The patient recovered following a single dose of 600 mg praziquantel.