Jorge Leitão

Issues in design and analysis of a randomized clinical trial to assess the safety of dental amalgam restorations in children

The Casa Pia Study of the Health Effects of Dental Amalgams in Children is a randomized clinical trial designed to assess the safety of low-level mercury exposure from dental amalgam restorations in children. It is being carried out in 507 students (8 to 12 years of age at enrollment) of the Casa Pia school system in Lisbon, Portugal, by an interdisciplinary collaborative research team from the University of Washington (Seattle) and the University of Lisbon, with funding from the National Institute of Dental and Craniofacial Research. Since the goal of the trial is to assess the safety of a treatment currently in use, rather than the efficacy of an experimental treatment, unique design issues come into play. The requirements to identify as participants children who have extensive unmet dental treatment needs and who can be followed for 7 years after initial treatment are somewhat in conflict, since those with the most treatment needs are usually in lower socioeconomic categories and more difficult to track. The identification of a primary study outcome measure around which to design the trial is problematic, since there is little evidence to indicate how health effects from such low-level exposure would be manifested. The solution involves the use of multiple outcomes. Since there are concerns about safety, multiple interim comparisons over time between treatment groups are called for which, in conjunction with the use of multiple outcomes, require an extension of statistical methodology to meet this requirement. Ethical questions that have to be addressed include whether assent of the children participating is required or appropriate, and whether the director of the school system, who is the legal guardian for approximately 20% of the students who are wards of the state and live in school residences, should provide consent for such a large number of children. Approaches taken to address these and other design issues are described.

The Contribution of Dental Amalgam to Urinary Mercury Excretion in Children

Background Urinary mercury concentrations are widely used as a measure of mercury exposure from dental amalgam fillings. No studies have evaluated the relationship of these measures in a longitudinal context in children. Objective We evaluated urinary mercury in children 8–18 years of age in relation to number of amalgam surfaces and time since placement over a 7-year course of amalgam treatment. Methods Five hundred seven children, 8–10 years of age at baseline, participated in a clinical trial to evaluate the neurobehavioral effects of dental amalgam in children. Subjects were randomized to either dental amalgam or resin composite treatments. Urinary mercury and creatinine concentrations were measured at baseline and annually on all participants. Results Treatment groups were comparable in baseline urinary mercury concentration (~ 1.5 μg/L). Mean urinary mercury concentrations in the amalgam group increased to a peak of ~ 3.2 μg/L at year 2 and then declined to baseline levels by year 7 of follow-up. There was a strong, positive association between urinary mercury and both number of amalgam surfaces and time since placement. Girls had significantly higher mean urinary mercury concentrations than boys throughout the course of amalgam treatment. There were no differences by race in urinary mercury concentration associated with amalgam exposure. Conclusions Urinary mercury concentrations are highly correlated with both number of amalgam fillings and time since placement in children. Girls excrete significantly higher concentrations of mercury in the urine than boys with comparable treatment, suggesting possible sex-related differences in mercury handling and susceptibility to mercury toxicity.

The mef(A) Gene Predominates among Seven Macrolide Resistance Genes Identified in Gram-Negative Strains Representing 13 Genera, Isolated from Healthy Portuguese Children

ABSTRACT Of the 176 randomly selected, commensal, gram-negative bacteria isolated from healthy children with low exposure to antibiotics, 138 (78%) carried one or more of the seven macrolide resistance genes tested in this study. These isolates included 79 (91%) isolates from the oral cavity and 59 (66%) isolates from urine samples. The mef(A) gene, coding for an efflux protein, was found in 73 isolates (41%) and was the most frequently carried gene. The mef(A) gene could be transferred from the donors into a gram-positive E. faecalis recipient and a gram-negative Escherichia coli recipient. The erm(B) gene transferred and was maintained in the E. coli transconjugants but was found in 0 to 100% of the E. faecalis transconjugants tested, while the other five genes could be transferred only into the E. coli recipient. The individual macrolide resistance genes were identified in 3 to 12 new genera. Eight (10%) of the oral isolates and 30 (34%) of the urine isolates for which the MICs were 2 to >500 μg of erythromycin per ml did not hybridize with any of the seven genes and may carry novel macrolide resistance genes.

A longitudinal study of neurological soft signs from late childhood into early adulthood.

Neurological examination of children includes the screening for soft neurological signs (NSS). There is little knowledge about their evolution during adolescence, except that their lasting presence has been associated with developmental, psychological, and cognitive disorders.

AGE AND SEX DIFFERENCES IN NEUROBEHAVIORAL PERFORMANCE: A STUDY OF PORTUGUESE ELEMENTARY SCHOOL CHILDREN

In this study normative data were obtained on measures of IQ, visual/motor, motor and memory functions among Portuguese elementary school children. Subjects were 228 females and 275 males, 8.0 to 11.9 years of age, in grades 2 through 4, who participated in a dental study. Performance on all tests improved with increasing age. Females performed better in rote verbal learning, psychomotor speed, and speed of information processing. Males had higher scores on tests of visual learning, visual memory, and fine motor speed and coordination. Nonverbal IQ had a significant impact on all tests except motor speed and coordination. Results represent an initial attempt to evaluate Portuguese children in educational and medical settings.

Staphylococcus Efflux msr(A) Gene Characterized in Streptococcus, Enterococcus, Corynebacterium, and Pseudomonas Isolates

ABSTRACT The staphylococcal msr(A) gene, coding for a macrolide efflux protein, was identified in three new gram-positive genera and one gram-negative genus. These msr(A) genes shared 99 to 100% identity with each other and the staphylococcal gene. This study demonstrates that the msr(A) gene has a wider host range than previously reported.

Biomarkers of kidney integrity in children and adolescents with dental amalgam mercury exposure: Findings from the Casa Pia children's amalgam trial

Mercury is toxic to the kidney, and dental amalgam is a source of mercury exposure. Few studies have evaluated the effects of dental amalgam on kidney function in a longitudinal context in children. Here, we evaluated urinary concentrations of glutathione S-transferases (GSTs) alpha and pi as biomarkers of renal proximal and distal tubular integrity, respectively, and albumin as a biomarker of glomerular integrity in children and adolescents 8-18 years of age over a 7-year course of dental amalgam treatment. Five hundred seven children, 8-12 years of age at baseline, participated in a clinical trial to evaluate the neurobehavioral and renal effects of dental amalgam in children. Subjects were randomized to either dental amalgam or resin composite treatments. Urinary GSTs alpha and pi, albumin, and creatinine concentrations were measured at baseline and annually in all subjects. Results were evaluated using linear regression analysis. GST-alpha concentrations were similar between treatment groups and in each sex and race (white vs. non-white) group in each follow-up year. GST-pi levels tended upward over the course of follow-up by four- to six-fold. This increase was seen in all groups irrespective of the treatment, race, or gender. Females had GST-pi levels approximately twice those of males at all ages. Albumin concentrations were constant throughout the follow-up period and did not differ by treatment, although females had 39% higher albumin levels than males. Additionally, we found no significant effects of amalgam treatment on the proportion of children with microalbuminuria (>30 mg/g creatinine). These findings are relevant within the context of childrens health risk assessment as relates to the safety of mercury exposure from dental amalgam on kidney function. These data also provide normative values for sensitive indices of renal functional integrity that may serve in the evaluation of children and adolescents with renal disorders.

URINARY PORPHYRIN EXCRETION IN NORMAL CHILDREN AND ADOLESCENTS

BACKGROUND Urinary porphyrins are diagnostic of various metabolic disorders and xenobiotic exposures, but comprehensive normative data for urinary porphyrin concentrations in children are currently unavailable. METHODS Subjects were participants in a prospective, randomized, controlled clinical trial of dental materials safety, 8 to 12 y at inception, who were followed longitudinally for 7 y after baseline with an extensive battery of neurobehavioral, neurological, renal function and urinary porphyrin assessments. Porphyrins were quantified by HPLC. Linear regression analyses were used to measure associations of porphyrin levels with age and gender. RESULTS Mean concentrations, 95% confidence intervals, and 10th, 50th, and 90th percentiles for all 5 typically excreted urinary porphyrins are presented by year of age and by gender. Unadjusted urinary concentrations (microg/l) of all 5 porphyrins remained relatively constant throughout the age range of 8-18 y for both males and females. In contrast, creatinine-adjusted urinary porphyrin concentrations (microg/g) declined significantly throughout this age range in both genders. Boys had significantly higher pentacarboxyl- and copro-porphyrin levels compared with girls both before and after creatinine adjustment. CONCLUSIONS Normative longitudinal data provided herein may facilitate the clinical assessment of pediatric metabolic disorders and may be of particular relevance in evaluating porphyrin changes as a biological indicator of disease or xenobiotic exposures among children and adolescents.

Urinary Porphyrin Excretion in Children with Mercury Amalgam Treatment: Findings from the Casa Pia Children's Dental Amalgam Trial

Increases in the urinary concentrations of pentacarboxyl- and coproporphyrins and the appearance of the atypical precoproporphyrin have been defined in relation to mercury (Hg) body burden in animal studies, and this change in the porphyrin excretion pattern has been described as a biomarker of occupational Hg exposure and toxicity in adult human subjects. In the present studies, urinary porphyrins were determined in relation to Hg exposure in children and adolescents, 8–18 yr of age, over the 7-yr course of a clinical trial designed to evaluate the neurobehavioral and renal effects of dental amalgam in children. Subjects were randomized to either dental amalgam or composite resin treatments. Urinary porphyrins and creatinine concentrations were measured at baseline and annually in all subjects. Results were evaluated using linear regression analysis. No significant differences between treatment groups (amalgam versus composite) were found when comparing all subjects for any of the porphyrins of interest. However, incipent amalgam treatment-specific increases were observed in the mean concentrations of penta-, precopro- and coproporphyrins especially when the analyses were restricted to younger subjects (8 to 9 yr old at baseline), and these increases were most apparent during yr 2 through 3 of follow-up, the period of highest mercury exposure from amalgam treatment. Based on the mean number of amalgam fillings received by children in this group (17.8), the renal Hg concentration associated with incipient increases in urinary porphyrins was estimated to be approximately 2.7 μg/g renal cortex. This value corresponds to an observed mean urinary Hg concentration of 3.2 μg/g creatinine, which is approximately fivefold less than that at which renal damage from Hg exposure is estimated to occur in children. These findings are consistent with growing evidence supporting the sensitivity of urinary porphyrins as a biological indicator of subclinical Hg exposure in children.

On the Structural Diversity of Sialoliths

Sialoliths from parotid and submaxillar glands have been characterized. Fractured and polished surfaces revealed an intrinsic structural diversity across the calculi sections. In general, the calculi presented highly mineralized amorphous-looking cores surrounded by concentric alternating mineralized and organic layers. The thickness of these layers decreased from the outer regions toward the center of the sialolith, illustrating a sequence of growth stages. Nevertheless, a significant variability could be detected among the specimens. In some cases, the calculi displayed multiple cores and lacked concentric laminated structures. In other instances, the specimens exhibited extensive regions of globular structures. In these cases, the globule diameter decreased across the radius toward the center of the sialoliths, and the globular structures tended to reorganize, forming bright and dark laminated layers surrounding the core. The participation of globular structures in the layer formation process points to morphogenetic mechanisms not previously described.