[Thyroid nodules in children: risk of malignancy for each Bethesda category]. / Nódulo tiroideo en pediatría: riesgo de malignidad para cada categoría Bethesda.

PURPOUSE: Thyroid nodules are unusual findings in children. Some authors highlight the higher rate of malignancy in this group. The Bethesda system, created in adults to stratify thyroid nodules according to the risk of malignancy, constitutes a reference system for the management of this pathology. The American Thyroid Association promotes its use in the pediatric population, although there is no available data showing an equivalent risk. The aim of this study was to assess the risk of malignancy represented by each Bethesda stage in a pediatric study population. METHODS: A retrospective cohort study was performed in pediatric patients with thyroid nodules biopsied by fine needle aspiration, during the period 2005-2017. During the follow-up, the outcome was assessed by comparing the Bethesda stage (cytology) with the surgical specimen histology. For patients not surgically treated, Bethesda Class was compared with the clinical and imaging follow up. RESULTS: 105 patients with fine needle aspiration of a thyroid nodule were analyzed and classified by the Bethesda system. 47 patients were excluded for incomplete follow-up. All Bethesda I nodules were benign, 6.6% of Bethesda II were malignant and all Bethesda IV, V and VI nodules were histologically malignant. CONCLUSION: The rate of malignancy among patients with Bethesda II, IV, V and VI was higher than published in Bethesda classification for adults. The risk of malignancy in pediatric patients might be greater than expected. These results may have a significant impact on follow-up strategies and also in therapeutic algorithms.

OBJETIVO: El nódulo tiroideo es un hallazgo infrecuente en pediatría. Algunos autores destacan la mayor tasa de malignidad en este grupo. La clasificación Bethesda, creada en pacientes adultos para estratificar los nódulos tiroideos según el riesgo de malignidad, constituye un sistema de referencia en el algoritmo terapéutico de esta patología. La American Thyroid Association propone homologar esta clasificación a la población pediátrica, si bien no existen datos que demuestren que el riesgo sea equivalente. El objetivo fue evaluar el riesgo de malignidad de cada categoría Bethesda en la población pediátrica. MATERIALES Y METODO: Se realizó un estudio de cohorte retrospectivo en pacientes pediátricos con nódulos tiroideos punzados con aguja fina durante el periodo 2005-2017. Luego se analizó su evolución ulterior comparando la categoría Bethesda asignada (citología) con la histología de la pieza quirúrgica. Para los pacientes sin indicación quirúrgica, se comparó con la evolución clínica e imagenológica. RESULTADOS: 105 pacientes cumplieron los criterios de inclusión. Se excluyeron 47 por seguimiento inadecuado. El 100% de los Bethesda I fueron nódulos benignos. El 6,5% de los Bethesda II fueron malignos. Todos los nódulos Bethesda IV, V y VI fueron malignos por histología. CONCLUSION: El porcentaje de malignidad entre los nódulos Bethesda II, IV, V y VI fue mayor al publicado. El riesgo de malignidad entre los pacientes pediátricos podría ser mayor al descrito en adultos para cada categoría de Bethesda. Estos resultados podrían ser significativos a la hora de establecer las estrategias tanto terapéuticas como de seguimiento.

An overview of 20 years of studies on the prevalence of human enteric viruses in shellfish from Galicia, Spain.

Galicia (NW Spain) has 1490 km of coastline, and its particular topography, characterized by the presence of fiord-like inlets, called rías, with an important primary production, makes this region very favourable for shellfish growth and culture. In fact, Galicia is one of the most important mussel producers in the world. Due to its proximity to cities and villages and the anthropogenic activities in these estuaries, and despite the routine official controls on the bivalve harvesting areas, contamination with material of faecal origin is sometimes possible but, current regulation based on Escherichia coli as an indicator micro-organism has been revealed as useful for bacterial contaminants, this is not the case for enteric viruses. The aim of this review is to offer a picture on the situation of different harvesting areas in Galicia, from a virological standpoint. A recompilation of results obtained in the last 20 years is presented, including not only the data for the well-known agents norovirus (NoV) and hepatitis A virus (HAV) but also data on emerging viral hazards, including sapovirus (SaV), hepatitis E virus (HEV) and aichivirus (AiV). Epidemiological differences related to diverse characteristics of the harvesting areas, viral genotype distribution or epidemiological links between environmental and clinical strains will also be presented and discussed. The presentation of these historical data all together could be useful for future decisions by competent authorities for a better management of shellfish growing areas.

Low prevalence of Aichi virus in molluscan shellfish samples from Galicia (NW Spain).

AIMS: The aim of this study was to detect and quantify Aichi virus (AiV) in shellfish from three estuaries in Galicia, the main producer of molluscs in Europe. METHODS AND RESULTS: A total of 249 shellfish samples were analysed using a reverse transcription-quantitative PCR procedure. AiV was detected in 15 of 249 (6·02%) samples. Ría de Ares-Betanzos showed the highest prevalence (11·1%), followed by Ría do Burgo (3·7%) and Ría de Vigo, (2·56%). AiV quantifications ranged from nonquantifiable (under the limit of quantification of the method) to 6·9 × 103 RNAc per g DT, with a mean value of 1·9 × 102 RNAc per g DT. CONCLUSION: Results obtained indicated that the prevalence of this enteric virus in the studied area is considerably lower than those of other enteric viruses, such as Norovirus, Sapovirus, HAV or HEV. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that detects the presence of AiV in shellfish from authorized harvesting areas in Spain. Further studies with clinical samples are needed to determine the potential risk of AiV for human health in Galicia.

Presence of multidrug-resistant enteric bacteria in dairy farm topsoil.

In addition to human and veterinary medicine, antibiotics are extensively used in agricultural settings, such as for treatment of infections, growth enhancement, and prophylaxis in food animals, leading to selection of drug and multidrug-resistant bacteria. To help circumvent the problem of bacterial antibiotic resistance, it is first necessary to understand the scope of the problem. However, it is not fully understood how widespread antibiotic-resistant bacteria are in agricultural settings. The lack of such surveillance data is especially evident in dairy farm environments, such as soil. It is also unknown to what extent various physiological modulators, such as salicylate, a component of aspirin and known model modulator of multiple antibiotic resistance (mar) genes, influence bacterial multi-drug resistance. We isolated and identified enteric soil bacteria from local dairy farms within Roosevelt County, NM, determined the resistance profiles to antibiotics associated with mar, such as chloramphenicol, nalidixic acid, penicillin G, and tetracycline. We then purified and characterized plasmid DNA and detected mar phenotypic activity. The minimal inhibitory concentrations (MIC) of antibiotics for the isolates ranged from 6 to >50 microg/mL for chloramphenicol, 2 to 8 microg/mL for nalidixic acid, 25 to >300 microg/mL for penicillin G, and 1 to >80 microg/mL for tetracycline. On the other hand, many of the isolates had significantly enhanced MIC for the same antibiotics in the presence of 5 mM salicylate. Plasmid DNA extracted from 12 randomly chosen isolates ranged in size from 6 to 12.5 kb and, in several cases, conferred resistance to chloramphenicol and penicillin G. It is concluded that enteric bacteria from dairy farm topsoil are multidrug resistant and harbor antibiotic-resistance plasmids. A role for dairy topsoil in zoonoses is suggested, implicating this environment as a reservoir for development of bacterial resistance against clinically relevant antibiotics.

Altered sugar selection and transport conferred by spontaneous point and deletion mutations in the lactose carrier of Escherichia coli.

Spontaneous mutants harboring the lacY gene on an F'-factor were isolated. Those mutants that failed to grow on 5 mM lactose minimal media plates were chosen for further study. The mutants showed striking mutations in the lactose carrier as well as in sugar selection properties during transport assays. DNA sequencing of the lacY gene of the mutants revealed the following mutations: M-1-I, R-144-W, G-370-C and a deletion of residues 387-392, located in helix 12 of the carrier. Transport studies indicated that ONPG transport ranged between 8 and 25% of normal for the M-1-I, G-370-C and D387-392 mutants and 51% of normal for the R-144-W mutant. The downhill transport of lactose was 2-fold greater than for melibiose in cells harboring the M-1-I mutation and 3-fold higher for cells with the G-370-C mutation. On the other hand, cells with the D387-392-deletion mutation showed no lactose downhill transport, but 47% melibiose transport. Accumulation of TMG, a lactose analog, was 3-fold higher than the accumulation of melibiose in cells with the G-370-C mutation. On the other hand, in cells with the D387-392 mutation, TMG accumulation was completely defective, whereas melibiose accumulation was 50-fold higher than that of TMG, indicating that one or more of these residues in helix 12 of the carrier play a role in the active transport of b-galactoside, but not a-galactoside sugars. Initial lactose downhill transport rates were too unreliable to obtain trustworthy kinetic data. TMG and melibiose accumulation activities were present, but severely reduced in the mutant containing the R144W mutation, confirming that Arg-144 is important for active transport. All transport data were normalized for expression levels. The results indicate that the affected residues play a role in dictating sugar specificity and transport in the lactose carrier. The results here are novel in that they represent mutations in unique locations along the lactose carrier protein. For example, the M-1-I mutation was located at the N-terminal cytoplasmic tail of the carrier. Furthermore, G-370-C was located in the periplasmic loop between helices 11 and 12, suggesting a role for residues in this loop in mediating sugar selection.

Mutants of the lactose carrier of Escherichia coli which show altered sugar recognition plus a severe defect in sugar accumulation.

Lactose and melibiose are actively accumulated by the wild-type Escherichia coli lactose carrier, which is an integral membrane protein energized by the proton motive force. Mutants of the E. coli lactose carrier were isolated by their ability to grow on minimal plates with succinate plus IPTG in the presence of the toxic lactose analog beta-thio-o-nitrophenylgalactoside (TONPG). TONPG-resistant mutants were streaked on melibiose MacConkey indicator plates, and red clones were picked. These melibiose positive mutants were then streaked on lactose MacConkey plates, and white clones were picked. Transport assays indicated that the mutants had altered sugar recognition and a defect in sugar accumulation. The mutants had a poor apparent K(m) for both lactose and melibiose in transport. One mutant had almost no ability to take up lactose, but melibiose downhill transport was 58% (V(max)) of normal. All of the mutants accumulated methyl-alpha-d-galactopyranoside (TMG) to only 8% or less of normal, and two failed to accumulate. Immunoblot analysis of the mutant lactose carrier proteins indicated that loss of sugar transport activity was not due to loss of expression in the membrane. Nucleotide sequencing of the lacY gene from the mutants revealed changes in the following amino acids of the lactose carrier: M23I, W151L, G257D, A295D and G377V. Two of the mutants (G257D and G377V) are novel in that they represent the first amino acids in periplasmic loops to be implicated with changes in sugar recognition. We conclude that the amino acids M23, W151, G257, A295 and G377 of the E. coli lactose carrier play either a direct or an indirect role in sugar recognition and accumulation.

Lactose carrier mutants of Escherichia coli with changes in sugar recognition (lactose versus melibiose).

The purpose of this research was to identify amino acid residues that mediate substrate recognition in the lactose carrier of Escherichia coli. The lactose carrier transports the alpha-galactoside sugar melibiose as well as the beta-galactoside sugar lactose. Mutants from cells containing the lac genes on an F factor were selected by the ability to grow on succinate in the presence of the toxic galactoside beta-thio-o-nitrophenylgalactoside. Mutants that grew on melibiose minimal plates but failed to grow on lactose minimal plates were picked. In sugar transport assays, mutant cells showed the striking result of having low levels of lactose downhill transport but high levels of melibiose downhill transport. Accumulation (uphill) of melibiose was completely defective in all of the mutants. Kinetic analysis of melibiose transport in the mutants showed either no change or a greater than normal apparent affinity for melibiose. PCR was used to amplify the lacY DNA of each mutant, which was then sequenced by the Sanger method. The following six mutations were found in the lacY structural genes of individual mutants: Tyr-26-->Asp, Phe-27-->Tyr, Phe-29-->Leu, Asp-240-->Val, Leu-321-->Gln, and His-322-->Tyr. We conclude from these experiments that Tyr-26, Phe-27, Phe-29 (helix 1), Asp-240 (helix 7), Leu-321, and His-322 (helix 10) either directly or indirectly mediate sugar recognition in the lactose carrier of E. coli.

Physiological evidence for an interaction between Glu-325 and His-322 in the lactose carrier of Escherichia coli.

Site-directed mutagenesis and second-site suppressor analysis have proven to be useful approaches to examine the role of charged amino acids in the structure and function of the lactose carrier of Escherichia coli. A lactose carrier mutant Glu-325 --> Ser failed to ferment melibiose and showed white clones on melibiose MacConkey indicator plates. Several red revertants were isolated from these plates. Two of these revertants showed a double mutation, the original mutation (Glu-325 --> Ser) plus His-322 --> Asp. Seven revertants showed a second site mutation His-322 --> Asn. Although the second site revertants failed to accumulate sugars they do show more rapid uptake of melibiose into cells containing alpha-galactosidase than the original mutant Glu-325 --> Ser. The complete loss of transport activity due to the removal of the negative charge at 325 can be partially compensated for by the introduction of a new negative charge at 322. A site-directed double mutant His-322 --> Asn/Glu-325 --> Asn showed a greater rate of lactose uptake (Vmax) than either of the single mutants His-322 --> Asn or Glu 325 --> Asn. It was concluded that there is some type of physiological interaction (possibly a salt bridge) between His-322 and Glu-325.

Mutational analysis and molecular modelling of an amino acid sequence motif conserved in antiporters but not symporters in a transporter superfamily.

Elements of a 'G X8 G X3 G P X2 G G' amino acid sequence motif were conserved in the fifth predicted membrane-spanning domains of 31 antiporters, but none of 27 symporters or uniporters that together comprise a 'superfamily' of structurally, related transport proteins. Molecular modelling and mechanics predicted that the GP dipeptide of this motif bends the antiporters' fifth transmembrane helices, and that the repeating pattern of glycine residues forms a pocket, devoid of side chains, on the surface of these helices. The glycine residue in the motif's GP dipeptide was conserved in 90% of these antiporters with alanine being the only observed substitution. Replacement of the glycine residue of the GP dipeptide with alanine and serine reduced the level of tetracycline resistance conferred by TetA(C), a tetracycline/H+ antiporter, by 74 and 81%, respectively. All other substitutions totally abolished resistance to tetracycline. In contrast, replacement of the glycine residue of the GP dipeptide did not abolish increased susceptibility to cadmium, another phenotype conferred by TetA(C) independent of resistance to tetracycline. These results suggest that the glycine of the GP dipeptide is necessary for the tetracycline/H+ antiport activity of TetA(C), rather than its expression, stability, or general three-dimensional structure.

Nucleotide and deduced protein sequences of the class D tetracycline resistance determinant: relationship to other antimicrobial transport proteins.

The nucleotide sequence of the plasmid pRA1 gene encoding the TetA(D) tetracycline/H+ antiporter was determined. The deduced amino acid sequence was compared with those of other antimicrobial and antiseptic transporters. The deduced product of tetA(D) is a 41.1-kDa protein consisting of 394 amino acids comprising 12 membrane-spanning domains. Three classes of amino acid motifs found in TetA(D) are highly conserved in other transporters, implying that they participate in structures necessary for substrate recognition, binding, or translocation. A common mechanism of transport is suggested, with subtle sequence variations accounting for varied substrate specificities, modes of transport, and directions of transport.