Secretory-IgA binding to intestinal microbiota attenuates inflammatory reactions as the intestinal barrier of preterm infants matures.

Previous work has shown that Secretory-IgA (SIgA) binding to the intestinal microbiota is variable and may regulate host inflammatory bowel responses. Nevertheless, the impact of the SIgA functional binding to the microbiota remains largely unknown in preterm infants whose immature epithelial barriers make them particularly susceptible to inflammation. Here, we investigated SIgA binding to intestinal microbiota isolated from stools of preterm infants <33 weeks gestation with various levels of intestinal permeability. We found that SIgA binding to intestinal microbiota attenuates inflammatory reactions in preterm infants. We also observed a significant correlation between SIgA affinity to the microbiota and the infant's intestinal barrier maturation. Still, SIgA affinity was not associated with developing host defenses, such as the production of mucus and inflammatory calprotectin protein, but it depended on the microbiota shifts as the intestinal barrier matures. In conclusion, we reported an association between the SIgA functional binding to the microbiota and the maturity of the preterm infant's intestinal barrier, indicating that the pattern of SIgA coating is altered as the intestinal barrier matures.

Optimizing congenital cytomegalovirus detection by pool testing in saliva by a rapid molecular test.

Universal congenital cytomegalovirus (cCMV) screening in saliva is increasingly recommended. The aim of our study was to correlate the performance of a point-of-care rapid molecular test with CMV real time PCR (CMV RT-PCR) detection, using saliva pool-testing in newborns under a universal screening strategy. Saliva swabs were prospectively collected from newborns < 21 days old and tested by Alethia-LAMP-CMV assay in pools of 5 samples. In positive pools, subjects were tested individually and by saliva and urine CMV RT-PCR. A subset of negative pools were studied with both techniques and viral loads in whole blood were determined in positive patients. From 1,642 newborns included in 328 pools, 8 were confirmed by urine CMV RT-PCR, (cCMV prevalence 0,49%). The PPA and NNA of the pooled saliva Alethia-LAMP-CMV testing were 87,5% and 99,8% with a negative and positive predictive value of 99,9% and 77,7%, respectively. Two false positives were detected (0,12%). A subset of 17 negative pools (85 samples), studied by saliva CMV RT-PCR, showed 100% concordance.  Conclusion: CMV pool-testing using a rapid molecular test in saliva proved feasible when compared to PCR gold standards. This strategy could improve cost-effectiveness for cCMV universal neonatal screening, based on the low prevalence of the infection and could be a more affordable approach in less developed regions with reduced detection capacity. What is Known: • cCMV is the most frequent congenital infection and a leading nongenetic cause of sensorineural hearing loss and brain disease. • Universal screening could allow early detection of congenitally infected infants, improving clinical outcome. • Saliva PCR is the preferred and non-invasive test for newborn cCMV screening. What is New: • The feasibility of a universal cCMV screening by pool-testing in saliva using a rapid test in pools of 5 samples. • PPA and NPA were 87,5 and 99,8% compared to CMV PCR in urine. • This strategy could be relevant specially in LMIC where detection capacity is reduced and could improve cost-effectiveness. • cCMV prevalence in our center was 0,49%.

Characterization of Adherent-Invasive Escherichia coli (AIEC) Outer Membrane Proteins Provides Potential Molecular Markers to Screen Putative AIEC Strains.

Adherent-invasive E. coli (AIEC) is a pathotype associated with the etiopathogenesis of Crohn's disease (CD), albeit with an as-yet unclear role. The main pathogenic mechanisms described for AIEC are adherence to epithelial cells, invasion of epithelial cells, and survival and replication within macrophages. A few virulence factors have been described as participating directly in these phenotypes, most of which have been evaluated only in AIEC reference strains. To date, no molecular markers have been identified that can differentiate AIEC from other E. coli pathotypes, so these strains are currently identified based on the phenotypic characterization of their pathogenic mechanisms. The identification of putative AIEC molecular markers could be beneficial not only from the diagnostic point of view but could also help in better understanding the determinants of AIEC pathogenicity. The objective of this study was to identify molecular markers that contribute to the screening of AIEC strains. For this, we characterized outer membrane protein (OMP) profiles in a group of AIEC strains and compared them with the commensal E. coli HS strain. Notably, we found a set of OMPs that were present in the AIEC strains but absent in the HS strain. Moreover, we developed a PCR assay and performed phylogenomic analyses to determine the frequency and distribution of the genes coding for these OMPs in a larger collection of AIEC and other E. coli strains. As result, it was found that three genes (chuA, eefC, and fitA) are widely distributed and significantly correlated with AIEC strains, whereas they are infrequent in commensal and diarrheagenic E. coli strains (DEC). Additional studies are needed to validate these markers in diverse strain collections from different geographical regions, as well as investigate their possible role in AIEC pathogenicity.

Genomic Loads and Genotypes of Respiratory Syncytial Virus: Viral Factors during Lower Respiratory Tract Infection in Chilean Hospitalized Infants.

The clinical impact of viral factors (types and viral loads) during respiratory syncytial virus (RSV) infection is still controversial, especially regarding newly described genotypes. In this study, infants with RSV bronchiolitis were recruited to describe the association of these viral factors with severity of infection. RSV antigenic types, genotypes, and viral loads were determined from hospitalized patients at Hospital Roberto del Río, Santiago, Chile. Cases were characterized by demographic and clinical information, including days of lower respiratory symptoms and severity. A total of 86 patients were included: 49 moderate and 37 severe cases. During 2013, RSV-A was dominant (86%). RSV-B predominated in 2014 (92%). Phylogenetic analyses revealed circulation of GA2, Buenos Aires (BA), and Ontario (ON) genotypes. No association was observed between severity of infection and RSV group (p = 0.69) or genotype (p = 0.87). After a clinical categorization of duration of illness, higher RSV genomic loads were detected in infants evaluated earlier in their disease (p < 0.001) and also in infants evaluated later, but coursing a more severe infection (p = 0.04). Although types and genotypes did not associate with severity in our children, higher RSV genomic loads and delayed viral clearance in severe patients define a group that might benefit from new antiviral therapies.

[Etiologic diagnosis in meningitis and encephalitis molecular biology techniques]. / Diagnóstico etiológico en meningitis y encefalitis por técnicas de biología molecular.

INTRODUCTION: The aetiological study of infections of the central nervous system has traditionally been performed using bacterial cultures and, more recently, using polymerase chain reaction (PCR) for herpes simplex virus (HSV). Bacterial cultures may not have good performance, especially in the context of patients who have received antibiotics prior to sampling, and a request for HSV only by PCR reduces the information to only one aetiological agent. The aim of this study is to determine the infectious causes of meningitis and encephalitis, using traditional microbiology and molecular biology to improve the aetiological diagnosis of these diseases. PATIENTS AND METHOD: A prospective study was conducted on 19 patients with suspected meningitis, admitted to the Luis Calvo Mackenna Hospital in Santiago, Chile, from March 1, 2011 to March 30, 2012. After obtaining informed consent, the CSF samples underwent cytochemical study, conventional culture, multiplex PCR for the major producing bacterial meningitis (N. meningitidis, S. pneumoniae, H. influenzae), real-time single PCR for HSV-1 and 2, VZV, EBV, CMV, HHV-6 and enterovirus. Clinical and epidemiological data were also collected from the clinical records. RESULTS: Of the 19 patients analysed, 2 were diagnosed by conventional methods and 7 by adding molecular biology (increase to 37%). Three patients had meningitis due to S. pneumoniae, one due to Enterobacter cloacae, 2 patients meningoencephalitis HSV-1, and one VZV meningitis. CONCLUSIONS: The addition of PCR to conventional diagnostic methods in CNS infections increases the probability of finding the causal agent. This allows a more adequate, timely and rational management of the disease.

TleA, a Tsh-like autotransporter identified in a human enterotoxigenic Escherichia coli strain.

Enterotoxigenic Escherichia coli (ETEC), a leading cause of acute diarrhea, colonizes the intestine by means of adhesins. However, 15 to 50% of clinical isolates are negative for known adhesins, making it difficult to identify antigens for broad-coverage vaccines. The ETEC strain 1766a, obtained from a child with watery diarrhea in Chile, harbors the colonization factor CS23 but is negative for other known adhesins. One clone, derived from an ETEC 1766a genomic library (clone G10), did not produce CS23 yet was capable of adhering to Caco-2 cells. The goal of this study was to identify the gene responsible for this capacity. Random transposon-based mutagenesis allowed the identification of a 4,110-bp gene that codes for a homologue of the temperature-sensitive hemagglutinin (Tsh) autotransporter described in avian E. coli strains (97% identity, 90% coverage) and that is called TleA (Tsh-like ETEC autotransporter) herein. An isogenic ETEC 1766a strain with a tleA mutation showed an adhesion level similar to that of the wild-type strain, suggesting that the gene does not direct attachment to Caco-2 cells. However, expression of tleA conferred the capacity for adherence to nonadherent E. coli HB101. This effect coincided with the detection of TleA on the surface of nonpermeabilized bacteria, while, conversely, ETEC 1766a seems to secrete most of the produced autotransporter to the medium. On the other hand, TleA was capable of degrading bovine submaxillary mucin and leukocyte surface glycoproteins CD45 and P-selectin glycoprotein ligand 1 (PSGL-1). These results suggest that TleA promotes colonization of the intestinal epithelium and that it may modulate the host immune response.

Flagellin and GroEL mediates in vitro binding of an atypical enteropathogenic Escherichia coli to cellular fibronectin.

BACKGROUND: Enteropathogenic Escherichia coli (EPEC) is distinguished mainly by the presence of EPEC adherence factor plasmid (pEAF) in typical EPEC (tEPEC) and its absence in atypical EPEC (aEPEC). The initial adherence to the intestinal mucosa is complex and mediated by adhesins other than bundle-forming pilus, which is not produced by aEPEC. Extracellular matrix (ECM) proteins of eukaryotic cells are commonly recognized by bacterial adhesins. Therefore, binding to ECM proteins may facilitate colonization, invasion and/or signaling by intestinal pathogens. Previous studies from our group demonstrated that aEPEC O26:H11 (strain BA2103) showed high binding activity to fibronectin, not shared by its counterpart, aEPEC O26:HNM. RESULTS: In the present study, using mass spectrometry after fibronectin-associated immunoprecipitation, two proteins, flagellin (50 kDa) and GroEL (52 kDa), were identified and BA2103 binding ability to fibronectin was inhibited in the presence of anti-H11 and anti-GroEL sera, but not by either naïve rabbit or other unrelated sera. It was also observed that the presence of purified flagellin inhibits adhesion of BA2103 to cellular fibronectin in a dose-dependent manner. Additionally, BA2103 GroEL is similar to the same protein of uropathogenic E. coli. CONCLUSIONS: Our results suggest that flagellin may play a role in the in vitro interaction of BA2103 with cellular fibronectin, and GroEL can be an accessory protein in this process.

Identification of cell surface-exposed proteins involved in the fimbria-mediated adherence of enteroaggregative Escherichia coli to intestinal cells.

Fimbria-mediated adherence to the intestinal epithelia is a key step in enteroaggregative Escherichia coli (EAEC) pathogenesis. To date, four fimbriae have been described for EAEC; aggregative adherence fimbria II (AAF/II) is the most important adherence factor for EAEC prototype strain 042. Previously, we described results showing that extracellular matrix (ECM) components might be involved in the recognition of AAF/II fimbriae by intestinal cells. In this study, we sought to identify novel potential receptors on intestinal epithelial cells recognized by the AAF/II fimbriae. Purified AafA-dsc protein, the major subunit of AAF/II fimbriae, was incubated with a monolayer of T84 cells, cross-linked to the surface-exposed T84 cell proteins, and immunoprecipitated by using anti-AafA antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of cellular proteins bound to AafA-dsc protein identified laminin (previously recognized as a potential receptor for AAF/II) and cytokeratin 8 (CK8). Involvement of the major subunit of AAF/II fimbriae (AafA protein) in the binding to recombinant CK8 was confirmed by adherence assays with purified AAF/II fimbriae, AafA-dsc protein, and strain 042. Moreover, HEp-2 cells transfected with CK8 small interfering RNA (siRNA) showed reduced 042 adherence compared with cells transfected with scrambled siRNA as a control. Adherence of 042 to HEp-2 cells preincubated with antibodies against ECM proteins or CK8 was substantially reduced. Altogether, our results supported the idea of a role of CK8 as a potential receptor for EAEC.

Prevalence of TPMT and ITPA gene polymorphisms and effect on mercaptopurine dosage in Chilean children with acute lymphoblastic leukemia.

BACKGROUND: Mercaptopurine (6-MP) plays a pivotal role in treatment of childhood acute lymphoblastic leukemia (ALL); however, interindividual variability in toxicity of this drug due to genetic polymorphism in 6-MP metabolizing enzymes has been described. We determined the prevalence of the major genetic polymorphisms in 6-MP metabolizing enzymes in Chilean children with ALL. METHODS: 103 Chilean pediatric patients with a confirmed diagnosis of ALL were enrolled. DNA was isolated from whole blood and genetic polymorphism in thiopurine S-methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) coding genes were detected by polymorphism chain reaction-restriction fragment length (PCR-RFLP) assay. RESULTS: The total frequency of variant TPMT alleles was 8%. TPMT*2, TPMT*3A and TPMT*3B alleles were found in 0%, 7%, and 1% of patients, respectively. For ITPA, the frequency of P32T allele was 3%. We did not observe any homozygous variant for TPMT and ITPA alleles. We also analyzed a subgroup of 40 patients who completed the maintenance phase of ALL treatment, and we found that patients carrying a TPMT gene variant allele required a significantly lower median cumulative dosage and median daily dosage of 6-MP than patients carrying wild type alleles. CONCLUSION: TMPT genotyping appears an important tool to further optimize 6-MP treatment design in Chilean patients with ALL.

A metagenomic approach to unveil the association between fecal gut microbiota and short-chain fatty acids in diarrhea caused by diarrheagenic Escherichia coli in children.

Diarrheagenic Escherichia coli (DEC) is the main cause of diarrhea in children under five years old. The virulence of DEC is tightly regulated by environmental signals influenced by the gut microbiota and its metabolites. Short-chain fatty acids (SCFAs) are the main metabolic product of anaerobic fermentation in the gut, but their role in DEC diarrhea has not yet been established. In this study, we determine the levels of acetate, propionate, and butyrate in stool samples from children with diarrhea caused by DEC, and we identify bacteria from the fecal gut microbiota associated with the production of SCFAs. The microbiota and SCFAs levels in stool samples obtained from 40 children with diarrhea and 43 healthy children were determined by 16S rRNA gene sequencing and HPLC, respectively. Additionally, shotgun metagenomics was used to identify metagenome-assembled genomes (MAGs) in a subgroup of samples. The results showed significantly higher levels of all SCFAs tested in diarrheal samples than in healthy controls. The abundance of Streptococcus sp., Limosilactobacillus, Blautia, Escherichia, Bacteroides, Megamonas, and Roseburia was higher in the DEC group than in healthy individuals. Functional analysis of bacteria and their main metabolic pathways made it possible to identify species MAGs that could be responsible for the detected SCFAs levels in DEC-positive diarrhea. In conclusion, based on our results and published data, we suggest that SCFAs may be important in the crosstalk between the microbiota and DEC pathogens in the gut.

Molecular mechanisms that mediate colonization of Shiga toxin-producing Escherichia coli strains.

Shiga toxin-producing Escherichia coli (STEC) is a group of pathogens which cause gastrointestinal disease in humans and have been associated with numerous food-borne outbreaks worldwide. The intimin adhesin has been considered for many years to be the only colonization factor in these strains. However, the rapid progress in whole-genome sequencing of different STEC serotypes has accelerated the discovery of other adhesins (fimbrial and afimbrial), which have emerged as important contributors to the intestinal colonization occurring during STEC infection. This review summarizes recent progress to identify and characterize, at the molecular level, novel adhesion and colonization factors in STEC strains, with an emphasis on their contribution to virulence traits, their host-pathogen interactions, the regulatory mechanisms controlling their expression, and their role as targets eliciting immune responses in the host.

Bacteria from gut microbiota associated with diarrheal infections in children promote virulence of Shiga toxin-producing and enteroaggregative Escherichia coli pathotypes.

Background: Diarrheagenic E. coli (DEC) pathogenicity relies on the interaction of bacteria with the host's gut environment, which is regulated by the resident microbiota. Previously, we identified indicative bacterial species of gut microbiota in DEC-positive stool samples from children. Here, we evaluated the role of two indicative species, Citrobacter werkmanii (CW) and Escherichia albertii (EA), in the virulence of two DEC pathotypes, Shiga toxin-producing (STEC) and enteroaggregative (EAEC) Escherichia coli. Methods: We determined the effect of supernatants obtained from CW and EA cultures on the gene expression of STEC strain 86-24 and EAEC strain 042 by RNA-seq analysis. We evaluated IL-8 secretion from T84 cells infected with these DEC strains in the presence or absence of the supernatant from EA. The effect of the supernatant from EA on the growth and adherence of STEC and EAEC to cells was also evaluated. Finally, we studied the effect of the EA supernatant on the STEC-induced inflammation mediated by the long polar fimbriae (Lpf) in T84 cells and the expression of plasmid-encoded toxin (Pet) in EAEC. Results: RNA-seq analysis revealed that several virulence factors in STEC and EAEC were upregulated in the presence of supernatants from CW and EA. Interestingly, an increase in the secretion of IL-8 was observed in cells infected with STEC or EAEC in the presence of a supernatant from EA. Similar results were observed with the supernatants obtained from clinical strains of E. albertii. The supernatant from EA had no effect on the growth of STEC and EAEC, or on the ability of these DEC strains to adhere to cells. We found that Pet toxin in EAEC was upregulated in the presence of a supernatant from EA. In STEC, using mutant strains for Lpf fimbriae, our data suggested that these fimbriae might be participating in the increase in IL-8 induced by STEC in cells in the presence of a supernatant from EA. Conclusion: Supernatant obtained from an indicative species of DEC-positive diarrhea could modulate gene expression in STEC and EAEC, and IL-8 secretion induced by these bacteria. These data provide new insights into the effect of gut microbiota species in the pathogenicity of STEC and EAEC.

Long polar fimbriae of enterohemorrhagic Escherichia coli O157:H7 bind to extracellular matrix proteins.

Adherence to intestinal cells is a key process in infection caused by enterohemorrhagic Escherichia coli (EHEC). Several adhesion factors that mediate the binding of EHEC to intestinal cells have been described, but the receptors involved in their recognition are not fully characterized. Extracellular matrix (ECM) proteins might act as receptors involved in the recognition of enteric pathogens, including EHEC. In this study, we sought to characterize the binding of EHEC O157:H7 to ECM proteins commonly present in the intestine. We found that EHEC prototype strains as well as other clinical isolates adhered more abundantly to surfaces coated with fibronectin, laminin, and collagen IV. Further characterization of this phenotype, by using antiserum raised against the LpfA1 putative major fimbrial subunit and by addition of mannose, showed that a reduced binding of EHEC to ECM proteins was observed in a long polar fimbria (lpf) mutant. We also found that the two regulators, H-NS and Ler, had an effect in EHEC Lpf-mediated binding to ECM, supporting the roles of these tightly regulated fimbriae as adherence factors. Purified Lpf major subunit bound to all of the ECM proteins tested. Finally, increased bacterial adherence was observed when T84 cells, preincubated with ECM proteins, were infected with EHEC. Taken together, these findings suggest that the interaction of Lpf and ECM proteins contributes to the EHEC colonization of the gastrointestinal tract.

[Molecular detection of gastrointestinal pathogens among children under 5 years old with diarrhea in a hospital center for rotavirus sentinel surveillance in Chile]. / Detección molecular de patógenos entéricos en niños con diarrea en un hospital centinela de vigilancia de rotavirus en Chile.

BACKGROUND: Infectious diarrhea is still a major problem in public health, especially in children under 5 years of age. The identification of the etiologic agent is important for the clinical management of the diarrhea episode and, from the epidemiological point of view, to implement control measures. AIM: To determine the presence of gastrointestinal pathogens in children under five years of age with diarrhea in a Chilean rotavirus surveillance center. METHODS: Observational study in children under five years of age who were hospitalized for diarrhea at the Dr. Luis Calvo Mackenna Hospital from December 2015 to December 2019. Molecular detection was performed using the FilmArray gastrointestinal (FilmArray GI®) panel. RESULTS: We analyzed 493 diarrheal stool samples of children, 427 samples (87%) were positive and 66 samples (13%) were negative. Of positive samples, 174 samples (41%) and 253 samples (59%) were positive for one or more pathogen, respectively. In children under one year and the group between one and four years there was a predominance of infections caused by enteric virus. Rotavirus and norovirus were the most common virus in both age groups. The most frequent bacteria were EPEC (27%), C. difficile (17%), EAEC (14%) and Campylobacter (9%). In parasites, Giardia lamblia and Cryptosporidium were identified, in 3% and 1% of the total samples, respectively. CONCLUSIONS: The molecular detection system used allowed an increase in the detection of enteropathogens in children under five years of age. The information generated by this type of surveillance could help to characterize the episodes of diarrhea in the population and might be a tool to technically advise the authorities in the decision-making process for the implementation of control measures.

CYP3A5 and UGT1A9 Polymorphisms Influence Immunosuppressive Therapy in Pediatric Kidney Transplant Recipients.

Background: Tacrolimus (TAC) and mycophenolic acid (MPA) are the main immunosuppressive drugs used in pediatric kidney transplantation. Single nucleotide polymorphisms (SNPs) in metabolizing enzymes and transporters might influence plasma levels of these drugs. Herein, we sought to determine the influence of SNPs on CYP3A5, MRP2 and UGT1A9 genes in Chilean pediatric kidney recipients using TAC and MPA. Patients and Methods: A prospective study was performed on 104 pediatric kidney recipients that used TAC and MPA for immunosuppression. The median age at the time of transplantation was 8.1 years [Q1-Q3 4.5-11.6 years] and the main clinical diagnosis was a structural anomaly. In a subgroup of patients, a complete steroid withdrawal was made at day 7. The CYP3A5 polymorphism (ancestral allele *1; variant allele *3) was determined in the entire cohort, while MRP2 -24G > A, UGT1A9 -275T > A, and UGT1A9 -2152C > T polymorphisms were determined in 53 patients. Genotypes were associated with trough drug concentrations (C0), dose requirements normalized by weight (TAC-D mg/kg) or body surface (MPA-D mg/m2), trough levels normalized by dose requirements (C0/D), and area under the curve in 12 h normalized by dose requirements (AUC0-12h/D). Results: The frequencies of the variant alleles CYP3A5*3, MRP2-24A, UGT1A9-275A, and UGT1A9-2152T were 76.9, 22.1, 6.6, and 2.9%, respectively. AUC0-12h/TAC-D were 1.6-fold higher in CYP3A5*3/*3 patients than in CYP3A5*1 carriers (CYP3A5*1/*3 and CYP3A5*1/*1). When analyzing patients with steroid withdrawal, CYP3A5*3/*3 patients had 1.7-fold higher AUC0-12h/TAC-D than the other genotypes. Patients carrying the CYP3A5*3/*3 genotype had higher TAC-C0, lower TAC-D and higher TAC-C0/D, consistently in a 6-months follow-up. Creatinine clearance was stable during the follow-up, regardless of the genotype. No significant differences between MRP2 and UGT1A9 genotypes were observed in MPA-C0, MPA-D or MPA-C0/D. However, patients carrying the UGT1A9-275A allele had lower AUC0-12h/MPA-D than those carrying the UGT1A9-275T ancestral allele. Conclusions: These results support that CYP3A5 and UGT1A9 genotyping in pediatric recipients might be useful and advisable to guide TAC and MPA dosing and monitoring in children that undergo kidney transplantation.

Performance of SARS-CoV-2 rapid antigen test compared with real-time RT-PCR in asymptomatic individuals.

Screening, testing and contact tracing plays a pivotal role in control of the COVID-19 pandemic. To enable this it is necessary to increase the testing capacity. This study compared a SARS-CoV-2 rapid antigen test (RAT) and RT-PCR in 842 asymptomatic individuals from Tarapacá, Chile. A sensitivity of 69.86%, specificity of 99.61%, PPV of 94.44% and NPP of 97.22% with Ct values (Ct > 27) that were significantly higher among individuals with false-negative RAT were reported. These results support the fact that RAT might have a significant impact on the identification of asymptomatic carriers in areas that lack suitable laboratories to perform SARS-CoV-2 real-time RT-PCR diagnostics, or the results take more than 24-48 h, as well as zones with high traffic of individuals such as border/customs, airports, interregional bus, train stations or in any mass testing campaign requiring rapid results.

Optimizing RT-PCR detection of SARS-CoV-2 for developing countries using pool testing.

The global shortage of reagents and kits for nucleic acid extraction and molecular detection of SARS-CoV-2 requires new cost-effective strategies for the diagnosis of suspected COVID-19 cases, especially in countries that need to increase detection capacity. Pooled nucleic acid testing has been extensively used as a cost-effective strategy for HIV, HepB, HepC and influenza. Also, protocols dispensing of RNA extraction appears as an attractive option for detection of SARS-CoV-2. In this study, we found that pooling of 5 samples showed that CT variations were in the range of 1.0-4,5 units, with less likelihood of a false negative result. Results of the sample without nucleic acid ex-traction, was unsatisfactory, with a significant increase in CT values, and thus for risk of a false negative result. In conclusion, pooling nasopharyngeal samples with both automated and manual extraction proved reliable, and thus a potential efficient alternative for the diagnosis of suspected COVID-19 in developing countries.

Gut Microbiota-Metabolome Changes in Children With Diarrhea by Diarrheagenic E. coli.

Background: Diarrheagenic Escherichia coli (DEC) strains are a main cause of diarrhea worldwide in children under 5 years old. DEC virulence is strongly regulated by environmental conditions and metabolites produced by the gut microbiota in the intestinal tract. In this study, we evaluated changes in gut microbiota-metabolome in children with or without diarrhea produced by DEC pathotypes. Goal: To determine gut microbiota composition and metabolome in stool samples obtained from healthy children and children with diarrhea positive for DEC pathotypes. Methods: We analyzed a total of 16 age-paired stool samples: 8 diarrheal samples positive for one DEC pathotype and 8 stool samples from healthy children. To identify the microbiota composition, we sequenced the V3-V4 region of the 16S rRNA and determined operational phylogenetic units (OPU). OPU were then used to predict metabolic pathways using the PICRUSt2 software. The presence of metabolites in stool samples was determined by LC-MS. A correlation analysis was performed with the main genera from each group and main metabolites. Bacteria associated with variance of main metabolites were identified using the MIMOSA2 software. Results: DEC and healthy groups showed a statistically different microbiota composition. A decrease in Firmicutes together with an increase in Bacteroidetes and Proteobacteria was found in the DEC group compared to the healthy group. Metabolic pathway predictions based on microbiota diversity showed that pathways involved in histidine and L-ornithine metabolism were significantly different between groups. A total of 88 metabolites detected by LC-MS were included in the metabolome analysis. We found higher levels of histamine and lower levels of ornithine in DEC samples than in the healthy group. Histamine and L-ornithine were associated with a specific microbiota species and the corresponding metabolic pathways. Conclusion: Stool samples from healthy children and children positive for DEC displayed a differential metabolome and microbiota composition. A strong correlation between a gut microbiota species and certain metabolites, such as histamine and L-ornithine, was found in the DEC group. This information might be useful to identify mechanisms and signaling molecules involved in the crosstalk between microbiota and DEC pathotypes.