The pediatric gut bacteriome and virome in response to SARS-CoV-2 infection.

Introduction: Since the beginning of the SARS-CoV-2 pandemic in early 2020, it has been apparent that children were partially protected from both infection and the more severe forms of the disease. Many different mechanisms have been proposed to explain this phenomenon, including children's frequent exposure to other upper respiratory infections and vaccines, and which inflammatory cytokines they are more likely to produce in response to infection. Furthermore, given the presence of SARS-CoV-2 in the intestine and its ability to infect enterocytes, combined with the well described immunomodulatory capabilities of the microbiome, another potential contributing factor may be the presence of certain protective microbial members of the gut microbiota (GM). Methods: We performed shotgun metagenomic sequencing and profiled both the bacteriome and virome of the GM of pediatric SARS-CoV-2 patients compared to healthy, age-matched subjects. Results: We found that, while pediatric patients do share some pro-inflammatory microbial signatures with adult patients, they also possess a distinct microbial signature of protective bacteria previously found to be negatively correlated with SARS-CoV-2 infectivity and COVID-19 severity. COVID-19 was also associated with higher fecal Cytomegalovirus load, and with shifts in the relative abundances of bacteriophages in the GM. Furthermore, we address how the preventative treatment of COVID-19 patients with antibiotics, a common practice especially in the early days of the pandemic, affected the bacteriome and virome, as well as the abundances of antimicrobial resistance and virulence genes in these patients. Discussion: To our knowledge, this is the first study to address the bacteriome, virome, and resistome of pediatric patients in response to COVID-19 and to preventative antibiotics use.

Case Report: The impact of severe cryptosporidiosis on the gut microbiota of a pediatric patient with CD40L immunodeficiency.

Cryptosporidium parvum is a protozoan parasite and one of the leading causes of gastroenteritis in the world, primarily affecting very young children and immunocompromised patients. While infection is usually self-limiting, it can become chronic and even lethal in these vulnerable populations, in whom Cryptosporidium treatments are generally ineffective, due to their acting in concert with a functioning immune system. Here, we describe a case of chronic cryptosporidiosis in a European child with severe CD40L immunodeficiency infected with Cryptosporidium parvum of the IIa20G1 subgenotype, a lineage which has thus far only ever been described in the Middle East. After years of on-off treatment with conventional and non-conventional anti-parasitic drugs failed to clear parasitosis, we performed targeted metagenomics to observe the bacterial composition of the patient's gut microbiota (GM), and to evaluate fecal microbiota transplantation (FMT) as a potential treatment option. We found that C. parvum infection led to significant shifts in GM bacterial composition in our patient, with consequent shifts in predicted intestinal functional signatures consistent with a state of persistent inflammation. This, combined with the patient's poor prognosis and increasing parasitic burden despite many rounds of anti-parasitic drug treatments, made the patient a potential candidate for an experimental FMT procedure. Unfortunately, given the many comorbidities that were precipitated by the patient's immunodeficiency and chronic C. parvum infection, FMT was postponed in favor of more urgently necessary liver and bone marrow transplants. Tragically, after the first liver transplant failed, the patient lost his life before undergoing FMT and a second liver transplant. With this case report, we present the first description of how cryptosporidiosis can shape the gut microbiota of a pediatric patient with severe immunodeficiency. Finally, we discuss how both our results and the current scientific literature suggest that GM modulations, either by probiotics or FMT, can become novel treatment options for chronic Cryptosporidium infection and its consequent complications, especially in those patients who do not respond to the currently available anti-parasitic therapies.

Primary cerebral cystic echinococcosis in a child from Roman countryside: Source attribution and scoping review of cases from the literature.

BACKGROUND: Human cystic echinococcosis (CE) is a zoonotic parasitic infection caused by the larval stage of the species belonging to the Echinococcus granulosus sensu lato (s.l.) complex. Parasitic cysts causing human CE are mainly localized in the liver and in the lungs. In a smaller number of cases, larvae may establish in any organ or tissue, including the central nervous system (CNS). Cerebral CE (CCE) is rare but poses serious clinical challenges. METHODS: This study presents a case of CCE in a child living in the countryside near Rome (Italy), along with a comparative molecular analysis of the isolated cyst specimens from the patient and sheep of local farms. We also systematically searched the literature to summarize the most relevant epidemiological and clinical aspects of this uncommon localization. FINDINGS: The comparative molecular analysis confirmed that the infection was caused by E. granulosus sensu stricto (s.s.) (G3 genotype), and most likely acquired in the family farm. The literature search identified 2,238 cases of CCE. In 80.51% of cases, brain was the only localization and single CCE cysts were present in 84.07% of cases. Mean patients' age was 20 years and 70.46% were children. Cyst rupture was reported in 12.96% and recurrence of CCE after treatment in 9.61% of cases. Permanent disability was reported in 7.86% of cases, while death occurred in 6.21%. In case series reporting all CE localization, CCE represented 1.5% of all CE cases. In the few reports that identified at molecular level the CCE cyst, E. granulosus s.s. was found in 40% and E. canadensis in 60% of cases. CONCLUSIONS: We report a rare case of CCE and evidenced the probable local origin of infection. The proportions of CE cases with uncommon localizations and with high impact on patients' lives have been globally neglected and should be included in the computation of the global burden of CE.

Gut Microbiota Ecological and Functional Modulation in Post-Stroke Recovery Patients: An Italian Study.

Ischemic stroke (IS) can be caused by perturbations of the gut-brain axis. An imbalance in the gut microbiota (GM), or dysbiosis, may be linked to several IS risk factors and can influence the brain through the production of different metabolites, such as short-chain fatty acids (SCFAs), indole and derivatives. This study examines ecological changes in the GM and its metabolic activities after stroke. Fecal samples of 10 IS patients were compared to 21 healthy controls (CTRLs). GM ecological profiles were generated via 16S rRNA taxonomy as functional profiles using metabolomics analysis performed with a gas chromatograph coupled to a mass spectrometer (GC-MS). Additionally fecal zonulin, a marker of gut permeability, was measured using an enzyme-linked immuno assay (ELISA). Data were analyzed using univariate and multivariate statistical analyses and correlated with clinical features and biochemical variables using correlation and nonparametric tests. Metabolomic analyses, carried out on a subject subgroup, revealed a high concentration of fecal metabolites, such as SCFAs, in the GM of IS patients, which was corroborated by the enrichment of SCFA-producing bacterial genera such as Bacteroides, Christensellaceae, Alistipes and Akkermansia. Conversely, indole and 3-methyl indole (skatole) decreased compared to a subset of six CTRLs. This study illustrates how IS might affect the gut microbial milieu and may suggest potential microbial and metabolic biomarkers of IS. Expanded populations of Akkermansia and enrichment of acetic acid could be considered potential disease phenotype signatures.

The metaproteome of the gut microbiota in pediatric patients affected by COVID-19.

Introduction: The gut microbiota (GM) play a significant role in the infectivity and severity of COVID-19 infection. However, the available literature primarily focuses on adult patients and it is known that the microbiota undergoes changes throughout the lifespan, with significant alterations occurring during infancy and subsequently stabilizing during adulthood. Moreover, children have exhibited milder symptoms of COVID-19 disease, which has been associated with the abundance of certain protective bacteria. Here, we examine the metaproteome of pediatric patients to uncover the biological mechanisms that underlie this protective effect of the GM. Methods: We performed nanoliquid chromatography coupled with tandem mass spectrometry on a high resolution analytical platform, resulting in label free quantification of bacterial protein groups (PGs), along with functional annotations via COG and KEGG databases by MetaLab-MAG. Additionally, taxonomic assignment was possible through the use of the lowest common ancestor algorithm provided by Unipept software. Results: A COVID-19 GM functional dissimilarity respect to healthy subjects was identified by univariate analysis. The alteration in COVID-19 GM function is primarily based on bacterial pathways that predominantly involve metabolic processes, such as those related to tryptophan, butanoate, fatty acid, and bile acid biosynthesis, as well as antibiotic resistance and virulence. Discussion: These findings highlight the mechanisms by which the pediatric GM could contribute to protection against the more severe manifestations of the disease in children. Uncovering these mechanisms can, therefore, have important implications in the discovery of novel adjuvant therapies for severe COVID-19.

The Relationship Between Pediatric Gut Microbiota and SARS-CoV-2 Infection.

This is the first study on gut microbiota (GM) in children affected by coronavirus disease 2019 (COVID-19). Stool samples from 88 patients with suspected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and 95 healthy subjects were collected (admission: 3-7 days, discharge) to study GM profile by 16S rRNA gene sequencing and relationship to disease severity. The study group was divided in COVID-19 (68), Non-COVID-19 (16), and MIS-C (multisystem inflammatory syndrome in children) (4). Correlations among GM ecology, predicted functions, multiple machine learning (ML) models, and inflammatory response were provided for COVID-19 and Non-COVID-19 cohorts. The GM of COVID-19 cohort resulted as dysbiotic, with the lowest α-diversity compared with Non-COVID-19 and CTRLs and by a specific ß-diversity. Its profile appeared enriched in Faecalibacterium, Fusobacterium, and Neisseria and reduced in Bifidobacterium, Blautia, Ruminococcus, Collinsella, Coprococcus, Eggerthella, and Akkermansia, compared with CTRLs (p < 0.05). All GM paired-comparisons disclosed comparable results through all time points. The comparison between COVID-19 and Non-COVID-19 cohorts highlighted a reduction of Abiotrophia in the COVID-19 cohort (p < 0.05). The GM of MIS-C cohort was characterized by an increase of Veillonella, Clostridium, Dialister, Ruminococcus, and Streptococcus and a decrease of Bifidobacterium, Blautia, Granulicatella, and Prevotella, compared with CTRLs. Stratifying for disease severity, the GM associated to "moderate" COVID-19 was characterized by lower α-diversity compared with "mild" and "asymptomatic" and by a GM profile deprived in Neisseria, Lachnospira, Streptococcus, and Prevotella and enriched in Dialister, Acidaminococcus, Oscillospora, Ruminococcus, Clostridium, Alistipes, and Bacteroides. The ML models identified Staphylococcus, Anaerostipes, Faecalibacterium, Dorea, Dialister, Streptococcus, Roseburia, Haemophilus, Granulicatella, Gemmiger, Lachnospira, Corynebacterium, Prevotella, Bilophila, Phascolarctobacterium, Oscillospira, and Veillonella as microbial markers of COVID-19. The KEGG ortholog (KO)-based prediction of GM functional profile highlighted 28 and 39 KO-associated pathways to COVID-19 and CTRLs, respectively. Finally, Bacteroides and Sutterella correlated with proinflammatory cytokines regardless disease severity. Unlike adult GM profiles, Faecalibacterium was a specific marker of pediatric COVID-19 GM. The durable modification of patients' GM profile suggested a prompt GM quenching response to SARS-CoV-2 infection since the first symptoms. Faecalibacterium and reduced fatty acid and amino acid degradation were proposed as specific COVID-19 disease traits, possibly associated to restrained severity of SARS-CoV-2-infected children. Altogether, this evidence provides a characterization of the pediatric COVID-19-related GM.

Auricular leishmaniasis in a child successfully treated with intralesional amphotericin B.

Cutaneous leishmaniasis (CL) is the most frequent form of leishmaniasis. The auricle is an extremely rare site for CL in the Old World. Auricular CL may be mistaken for other entities, such as relapsing polychondritis (RP). Here we report a pediatric case of Old World auricular CL mimicking RP in a child successfully treated with intralesional liposomal amphotericin B.

Cryptosporidium: Still Open Scenarios.

Cryptosporidiosis is increasingly identified as a leading cause of childhood diarrhea and malnutrition in both low-income and high-income countries. The strong impact on public health in epidemic scenarios makes it increasingly essential to identify the sources of infection and understand the transmission routes in order to apply the right prevention or treatment protocols. The objective of this literature review was to present an overview of the current state of human cryptosporidiosis, reviewing risk factors, discussing advances in the drug treatment and epidemiology, and emphasizing the need to identify a government system for reporting diagnosed cases, hitherto undervalued.

What's in a child's ear? A case of otomyiasis by Sarcophaga argyrostoma (Diptera, Sarcophagidae).

A clinical report of otomyiasis in a 1-year-old girl is reported. A III instar larva of Sarcophaga sp. was microscopically identified and Sarcophaga (Liopygia) argyrostoma (Diptera, Sarcophagidae) was suspected. A molecular method targeting a fragment of the cox1 gene was used to confirm the identity of the specimen. Although myiases are not frequent manifestations in otolaryngology, they should arouse the attention of doctors, social workers and parents dealing with disabled people, the elderly and children. This contribution also highlights the need of combining microscopy and molecular tools to achieve a correct and reliable identification of the specimen/s.

Clinical Parasitology and Parasitome Maps as Old and New Tools to Improve Clinical Microbiomics.

A growing body of evidence shows that dysbiotic gut microbiota may correlate with a wide range of disorders; hence, the clinical use of microbiota maps and fecal microbiota transplantation (FMT) can be exploited in the clinic of some infectious diseases. Through direct or indirect ecological and functional competition, FMT may stimulate decolonization of pathogens or opportunistic pathogens, modulating immune response and colonic inflammation, and restoring intestinal homeostasis, which reduces host damage. Herein, we discuss how diagnostic parasitology may contribute to designing clinical metagenomic pipelines and FMT programs, especially in pediatric subjects. The consequences of more specialized diagnostics in the context of gut microbiota communities may improve the clinical parasitology and extend its applications to the prevention and treatment of several communicable and even noncommunicable disorders.

Strongyloides stercoralis Infestation in a Child: How a Nematode Can Affect Gut Microbiota.

Background: Strongyloidiasis is a neglected tropical disease caused by the intestinal nematode Strongyloides stercoralis and characterized by gastrointestinal and pulmonary involvement. We report a pediatric case of strongyloidiasis to underline the response of the host microbiota to the perturbation induced by the nematode. Methods: We performed a 16S rRNA-metagenomic analysis of the gut microbiota of a 7-year-old female during and after S. stercolaris infection, investigating three time-point of stool samples' ecology: T0- during parasite infection, T1- a month after parasite infection, and T2- two months after parasite infection. Targeted-metagenomics were used to investigate ecology and to predict the functional pathways of the gut microbiota. Results: an increase in the alpha-diversity indices in T0-T1 samples was observed compared to T2 and healthy controls (CTRLs). Beta-diversity analysis showed a shift in the relative abundance of specific gut bacterial species from T0 to T2 samples. Moreover, the functional prediction of the targeted-metagenomics profiles suggested an enrichment of microbial glycan and carbohydrate metabolisms in the T0 sample compared with CTRLs. Conclusions: The herein report reinforces the literature suggestion of a putative direct or immune-mediated ability of S. stercolaris to promote the increase in bacterial diversity.

Mass Spectrometry Based-Proteomic Analysis of Anisakis spp.: A Preliminary Study towards a New Diagnostic Tool.

Anisakiasis is nowadays a well-known infection, mainly caused by the accidental ingestion of Anisakis larvae, following the consumption of raw or undercooked fishes and cephalopods. Due to the similarity of symptoms with those of common gastrointestinal disorders, this infection is often underestimated, and the need for new specific diagnostic tools is becoming crucial. Given the remarkable impact that MALDI-TOF MS biotyping had in the last decade in clinical routine practice for the recognition of bacterial and fungi strains, a similar scenario could be foreseen for the identification of parasites, such as nematodes. In this work, a MALDI-TOF MS profiling of Anisakis proteome was pursued with a view to constructing a first spectral library for the diagnosis of Anisakis infections. At the same time, a shotgun proteomics approach by LC-ESI-MS/MS was performed on the two main fractions obtained from protein extraction, to evaluate the protein species enriched by the protocol. A set of MALDI-TOF MS signals associated with proteins originating in the ribosomal fraction of the nematode extract was selected as a potential diagnostic tool for the identification of Anisakis spp.

Challenging diagnosis of congenital malaria in non-endemic areas.

BACKGROUND: Congenital malaria is usually defined as the detection of asexual forms of Plasmodium spp. in a blood sample of a neonate during perinatal age if there is no possibility of postpartum infection by a mosquito bite. The incidence of congenital malaria is highly variable and seems related to several factors, such as different diagnostic methods for Plasmodium spp. detection, and area in which the epidemiologic analyses are performed. In non-endemic countries, cases of congenital malaria are rare. Hereby, a case of a congenital malaria in an HIV exposed child is reported. CASE PRESENTATION: A 2-month-old male child was admitted to Bambino Gesù Children's Hospital due to anaemia and exposure to HIV. He was born prematurely in Italy by cesarean section at 34 weeks' gestation after a bicorial, biamniotic pregnancy by a migrant woman from Nigeria. He was the first of non-identical twins. Combined with anaemia, spleen and liver enlargement was noted, malaria was hypothesized. Malaria laboratory panel was performed on the newborn, mother and other twin blood samples, as follows: (i) malaria rapid diagnostic test (RDT); (ii) Giemsa-stained thick and thin blood smears for Plasmodium spp. identification and parasitaemia titration; (iii) molecular screening and typing of Plasmodium spp. by multiplex qualitative PCR assay based on 18S rRNA gene. Genotyping of Plasmodium falciparum isolates from mother and child was performed by neutral microsatellite and highly polymorphic marker amplification. CONCLUSIONS: The maternal RDT sample was negative, while the infant RDT was positive; in both cases microscopy of blood smears and PCR showed infection with P. falciparum. Two of the genotypic molecular markers displayed different allelic variants between the two samples. This difference could imply infection multiplicity of the mother during the pregnancy, possibly harbouring more than one isolate, only one of them being transmitted to the newborn while the other persisting in the mother's blood. Because of the increasing number of pregnant women coming from endemic areas for malaria, an accurate anamnesis of infant's mother, and the inclusion of Plasmodium spp. research into TORCH screenings for mother-infant pair at birth, aiming at reducing morbidity and mortality associated to the disease might be suitable.

Serological evaluation for Chagas disease in migrants from Latin American countries resident in Rome, Italy.

BACKGROUND: Chagas disease (CD) is a systemic parasitic infection caused by the protozoan Trypanosoma cruzi, whose chronic phase may lead to cardiac and intestinal disorders. Endemic in Latin America where it is transmitted mainly by vectors, large-scale migrations to other countries have turned CD into a global health problem because of its alternative transmission routes through blood transfusion, tissue transplantation, or congenital. Aim of this study was to compare the performance of two commercially available tests for serological diagnosis of CD in a group of Latin American migrants living in a non-endemic setting (Rome, Italy). The study was based on a cross-sectional analysis of seroprevalence in this group. Epidemiological risk factors associated to CD were also evaluated in this study population. METHODS: The present study was conducted on 368 subjects from the Latin American community resident in Rome. Following WHO guidelines, we employed a diagnostic strategy based on two tests to detect IgG antibodies against T. cruzi in the blood (a lysate antigen-based ELISA and a chemiluminescent microparticle CMIA composed of multiple recombinant antigens), followed by a third test (an immunochromatographic assay) on discordant samples. RESULTS: Our diagnostic approach produced 319/368 (86.7%) concordant negative and 30/368 (8.1%) concordant positive results after the first screening. Discrepancies were obtained for 19/368 (5.2%) samples that were tested using the third assay, obtaining 2 more positive and 17 negative results. The final count of positive samples was 32/368 (8.7% of the tested population). Increasing age, birth in Bolivia, and previous residence in a mud house were independent factors associated with T. cruzi positive serology. CONCLUSIONS: Serological diagnosis of CD is still challenging, because of the lack of a reference standard serological assay for diagnosis. Our results reaffirm the importance of performing CD screening in non-endemic countries; employing a fully automated and highly sensitive CMIA assay first could be a cost- and resource-effective strategy for mass screening of low-risk patients. However, our results also suggest that the WHO strategy of using two different serological assays, combined with epidemiological information, remains the best approach for patients coming from endemic countries.

Cystic echinococcosis in a single tertiary care center in Rome, Italy.

BACKGROUND: Cystic echinococcosis (CE) is a chronic, clinically complex, and neglected disease. Its prevalence in Italy, a country of medium to high endemicity, remains poorly defined, as notification has long ceased to be mandatory. METHODS: We set up a retrospective cohort study involving all CE patients followed at our institute between January 2005 and December 2012. Demographical and clinical features were recorded and analyzed. RESULTS: CE was found in 28 patients (64.3%), mostly Italians from the central regions (50%), followed by subjects from the islands (33.3%) and Southern Italy (16.7%). Their median age was 45 years (IQR: 38.5-66.5), with Eastern Europeans being significantly younger (28 years, IQR: 19-39) than other patients (P ≤ 0.0001). A total of 149 cysts, mostly with hepatic localization (96%), were described. Based on the WHO classification, the cysts were mainly small (80.5%) and active (CE1 (73.8%); CE2 (7.4%)). Active cysts were more common in Eastern Europeans (85.7%) than Italians (66.7%). CONCLUSION: Our data confirm CE occurrence in Italy. We emphasize the importance to have a national CE registry, opportunely recently introduced. This is essential to assess CE prevalence in this country, implement appropriate control measures, and improve patient management.

Accuracy of indirect haemagglutination and western blot assays for the detection of anti-Schistosoma antibodies in non-severe febrile patients in two Tanzanian hospitals.

BACKGROUND: The diagnosis of schistosomiasis is usually based on clinical data associated with the detection of eggs in stool, urine, and/or rectal and bladder biopsy specimens. However antibody detection can be useful to indicate Schistosoma infection in those for whom eggs cannot be demonstrated. The aim of this study was to assess the seroprevalence of schistosomiasis and to evaluate the accuracy of indirect haemagglutination (IHA) and Western blot (WB) assays for the detection of anti-Schistosoma antibodies in 2 peripheral hospitals of the United Republic of Tanzania. METHODS: Between February and March 2007 blood samples were collected from 297 non-severe febrile outpatients who attended Chake Chake Hospital, Pemba Island and Tosamaganga Hospital, Iringa region in Tanzania. The samples were processed for Schistosoma antibodies by IHA and WB assays in Italy. RESULTS: Two hundred and sixty-two of 297 patients were schistosomiasis antibody-positive by IHA (88.2%). Of 142 patients positive by IHA, only 22 (12.4%) cases were confirmed by WB assay. The WB assay confirmed all 35 negative cases previously identified by IHA. The seroprevalence of Schistosoma at Chake Chake Hospital was lower than in Tosamaganga Hospital (9/97, 9.3% vs 13/80, 16.2%). CONCLUSIONS: Schistosomiasis is endemic in Tanzania, being more prevalent on the mainland than on Pemba Island. The implications of this study are of public health relevance and suggest the need for increased efforts in large-scale chemotherapy-based morbidity control programmes, integrated with those for other soil-transmitted helminthiases, in these 2 peripheral areas of the United Republic of Tanzania.