Prechtl's method to assess general movements: Inter-rater reliability during the preterm period.

INTRODUCTION: Prechtl's method (GMA) is a test for the functional assessment of the young nervous system. It involves a global and a detailed assessment of the general movements (GMs) and has demonstrated validity. Data on the reliability of both assessments in the preterm period are scarce. This study aimed to evaluate the inter-rater reliability for the global and detailed assessments of the preterm writhing GMA. MATERIALS AND METHODS: The study participants were 69 infants born at <37 gestational weeks and admitted to the neonatal intensive care unit. They were randomly assigned to five pairs of raters. Raters assessed infants' GMs using preterm videos. Outcome variables were (a) the GMs classification (normal versus abnormal; normal versus abnormal subcategories) and (b) the general movements optimality score (GMOS), obtained through the global and detailed assessments. The Gwet's AC1 and the intraclass correlation coefficient (ICC) were calculated for the GMs classification and the GMOS, respectively. RESULTS: The global assessment presented an AC1 = 0.84 [95% CI = 0.54,1] for the GMs binary classification and an AC1 = 0.67 [95% CI = 0.38,0.89] for the GMs classification with abnormal subcategories. The detailed assessment presented an ICC = 0.72 [95% CI = 0.39,0.90] for the GMOS. CONCLUSIONS: Inter-rater reliability was high and substantial for the global assessment and good for the detailed assessment. However, the small sample size limited the precision of these estimates. Future research should involve larger samples of preterm infants to improve estimate precision. Challenging items such as assessing the neck and trunk, poor repertoire GMs, and tremulous movements may impact the preterm writhing GMA's inter-rater reliability. Therefore, ongoing training and calibration among raters is necessary. Further investigation in clinical settings can enhance our understanding of the preterm writhing GMA's reliability.

Infants prenatally exposed to SARS-CoV-2 show the absence of fidgety movements and are at higher risk for neurological disorders: A comparative study.

Congenital viral infections are believed to damage the developing neonatal brain. However, whether neonates exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) show manifestations of such damage remains unclear. For neurodevelopment evaluation, general movement assessments have been shown to be effective in identifying early indicators of neurological dysfunction, including the absence of fidgety movements. This study compared the early motor repertoire by general movement assessment at three to five months of age in neonates who were or were not prenatally exposed to SARS-CoV-2 to determine whether infants prenatally exposed to SARS-CoV-2 are at risk of developing neurological disorders. Fifty-six infants, including 28 in the exposed group of mothers without vaccination who had no need for intensive care and likely had SARS-CoV-2 infection close to the time of pregnancy resolution and 28 infants in the nonexposed group, were videotaped to compare their detailed early motor repertoires, in which a motor optimality score-revised (MOS-R) was calculated using Prechtl's method by using the chi-square or Mann-Whitney U tests. In the exposed group, 3 (11%) infants showed the absence of fidgety movements with a total MOS-R<14 points, and 3 (11%) other infants showed abnormal fidgety movements. Between groups, atypical body symmetry (p = 0.009) and MOS-R values were significantly lower (Z = -3.08, p = 0.002), with a large size effect (Cohen's d = 0.97). The consequences of this new virus go beyond the health of the pregnant mother, and these consequences in some of the infants in the exposed group are likely not transitory because of the absence of fidgety movements between 3-5 months; thus, these babies are at increased risk of developing a serious neurological disorder.

Different roles of conserved tyrosine residues of the acylated domains in folding and activity of RTX toxins.

Pore-forming repeats in toxins (RTX) are key virulence factors of many Gram-negative pathogens. We have recently shown that the aromatic side chain of the conserved tyrosine residue 940 within the acylated segment of the RTX adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in target cell membrane interaction of the toxin. Therefore, we used a truncated CyaA-derived RTX719 construct to analyze the impact of Y940 substitutions on functional folding of the acylated segment of CyaA. Size exclusion chromatography combined with CD spectroscopy revealed that replacement of the aromatic side chain of Y940 by the side chains of alanine or proline residues disrupted the calcium-dependent folding of RTX719 and led to self-aggregation of the otherwise soluble and monomeric protein. Intriguingly, corresponding alanine substitutions of the conserved Y642, Y643 and Y639 residues in the homologous RtxA, HlyA and ApxIA hemolysins from Kingella kingae, Escherichia coli and Actinobacillus pleuropneumoniae, affected the membrane insertion, pore-forming (hemolytic) and cytotoxic capacities of these toxins only marginally. Activities of these toxins were impaired only upon replacement of the conserved tyrosines  by proline residues. It appears, hence, that the critical role of the aromatic side chain of the Y940 residue is highly specific for the functional folding of the acylated domain of CyaA and determines its capacity to penetrate target cell membrane.

Almost half of the RTX domain is dispensable for complement receptor 3 binding and cell-invasive activity of the Bordetella adenylate cyclase toxin.

The whooping cough agent Bordetella pertussis secretes an adenylate cyclase toxin (CyaA) that through its large carboxy-proximal Repeat-in-ToXin (RTX) domain binds the complement receptor 3 (CR3). The RTX domain consists of five blocks (I-V) of characteristic glycine and aspartate-rich nonapeptides that fold into five Ca2+-loaded parallel ß-rolls. Previous work indicated that the CR3-binding structure comprises the interface of ß-rolls II and III. To test if further portions of the RTX domain contribute to CR3 binding, we generated a construct with the RTX block II/III interface (CyaA residues 1132-1294) linked directly to the C-terminal block V fragment bearing the folding scaffold (CyaA residues 1562-1681). Despite deletion of 267 internal residues of the RTX domain, the Ca2+-driven folding of the hybrid block III/V ß-roll still supported formation of the CR3-binding structure at the interface of ß-rolls II and III. Moreover, upon stabilization by N- and C-terminal flanking segments, the block III/V hybrid-comprising constructs competed with CyaA for CR3 binding and induced formation of CyaA toxin-neutralizing antibodies in mice. Finally, a truncated CyaAΔ1295-1561 toxin bound and penetrated erythrocytes and CR3-expressing cells, showing that the deleted portions of RTX blocks III, IV, and V (residues 1295-1561) were dispensable for CR3 binding and for toxin translocation across the target cell membrane. This suggests that almost a half of the RTX domain of CyaA is not involved in target cell interaction and rather serves the purpose of toxin secretion.

Cerebral Palsy: Early Markers of Clinical Phenotype and Functional Outcome.

The Prechtl General Movement Assessment (GMA) has become a cornerstone assessment in early identification of cerebral palsy (CP), particularly during the fidgety movement period at 3-5 months of age. Additionally, assessment of motor repertoire, such as antigravity movements and postural patterns, which form the Motor Optimality Score (MOS), may provide insight into an infant's later motor function. This study aimed to identify early specific markers for ambulation, gross motor function (using the Gross Motor Function Classification System, GMFCS), topography (unilateral, bilateral), and type (spastic, dyskinetic, ataxic, and hypotonic) of CP in a large worldwide cohort of 468 infants. We found that 95% of children with CP did not have fidgety movements, with 100% having non-optimal MOS. GMFCS level was strongly correlated to MOS. An MOS > 14 was most likely associated with GMFCS outcomes I or II, whereas GMFCS outcomes IV or V were hardly ever associated with an MOS > 8. A number of different movement patterns were associated with more severe functional impairment (GMFCS III-V), including atypical arching and persistent cramped-synchronized movements. Asymmetrical segmental movements were strongly associated with unilateral CP. Circular arm movements were associated with dyskinetic CP. This study demonstrated that use of the MOS contributes to understanding later CP prognosis, including early markers for type and severity.

Influence of the Co-Administration of Heptavalent Conjugate Vaccine PCV7-TT on the Immunological Response Elicited by VA-MENGOC-BC® and Heberpenta®-L in Rabbits.

Finlay Vaccine Institute is developing a new heptavalent conjugate vaccine against Streptococcus pneumoniae. As infants are the target population, PCV7-TT will be necessarily co-administered with other vaccines, and then, the interactions represent a concern. The aim of this work is to evaluate the possible immunological interferences in rabbits as animal experimental model. Rabbits were immunized with Heberpenta®-L, VA-MENGOC-BC®, and PCV7-TT. Blood samples were taken fourteen days after final immunization for obtaining sera. Antibody responses to all antigens were evaluated by indirect ELISA. Functional responses against diphtheria and tetanus toxoid were done by in vivo seroneutralization assay. No interference was observed by PCV7-TT over the humoral response against diphtheria toxoid and meningococcal antigens (p > 0.05). A nonstatistically significant reduction (p > 0.05) was observed in the case of the humoral response against Haemophilus influenzae type b oligosaccharide. Concomitant administration of Heberpenta®-L and PCV7-TT increased twice the antibody titers as well as the protective activity against tetanus toxoid, but no statistical differences were found. The co-administration did not induce a reduction in the percent of responders against pneumococcal polysaccharides contained in PCV7-TT vaccine. Concomitant administration of PCV7-TT did not induce interferences over the evaluated antigens of Heberpenta®-L and VA-MENGOC-BC®. Also, no interference was observed on the immune response elicited by PCV7-TT. These preclinical results suggest that PCV7-TT will not result in a serious problem over the immune response elicited by the licensed vaccines Heberpenta®-L and VA-MENGOC-BC®. However, the clinical interference could be strictly studied during clinical trials in infants.

Validación y aplicación de un ELISA para la cuantificación de anticuerpos IgG contra polisacárido capsular Vi de Salmonella Typhi / Validation and application of an ELISA for the quantification of IgG antibodies against Salmonella Typhi Vi capsular polysaccharide

Como parte de las etapas de investigación y desarrollo de un candidato vacunal conjugado contra Salmonella Typhi, se desarrolló un ELISA indirecto para la cuantificación de anticuerpos IgG contra el polisacárido Vi de esta bacteria. En este trabajo se presentan los resultados del proceso de validación, en el que se determinaron el intervalo y linealidad de la curva, la precisión intra e interensayo, la exactitud, la especificidad, el límite de detección y la robustez. La curva de calibración, generada con un suero estándar interno, presentó un buen ajuste a una función polinómica y un intervalo entre las diluciones 1/100 y 1/3200. Los coeficientes de variación en los ensayos de precisión y robustez y los porcentajes de recobrado estuvieron en los intervalos establecidos para cada uno (≤10 por ciento, ≤20 por ciento y 90-110 por ciento respectivamente). El ensayo presentó una especificidad óptima, obteniéndose señales de DO superiores a 1,3 para sueros positivos contra Vi y bajas para sueros contra antígenos no relacionados. Los resultados avalan el empleo de este ELISA cuantitativo en ensayos de inmunogenicidad para la liberación de lotes de conjugados de Vi. Igualmente, sustentan su uso para la evaluación de la inmunogenicidad de formulaciones de polisacárido Vi y conjugados de polisacárido Vi a proteínas en fases de investigación y desarrollo(AU)

An indirect ELISA for the quantification of IgG antibodies against the Vi polysaccharide of this bacteria was developed as a part of the stages of Research and Development of a conjugate vaccinal candidate against Salmonella Typhi. The results of the validation process are presented in this paper, in which the interval and linearity of the curve, the intra- and inter-assay precision, accuracy, specificity, limit of detection and robustness were determined. The calibration curve generated with an internal standard serum provided a good fit to a polynomial function and an interval between 1/100 and 1/3200 dilutions. The coefficients of variation in the precision and robustness tests and the percentages of recovery were in intervals established for each one (≤10 percent, ≤20 percent and 90-110 percent, respectively). The assay presented an optimal specificity, obtaining OD signals above 1.3 for positive sera against Vi and low for sera against unrelated antigens. The results support the use of this quantitative ELISA in immunogenicity assays for batch release of Vi conjugates. Likewise, they support their use for the immunogenicity evaluation of Vi polysaccharide formulations and Vi polysaccharide conjugates to proteins in phases of research and development(AU)