An anthropometric approach to characterising neonatal morbidity and body composition, using air displacement plethysmography as a criterion method.

BACKGROUND: With the greatest burden of infant undernutrition and morbidity in low and middle income countries (LMICs), there is a need for suitable approaches to monitor infants in a simple, low-cost and effective manner. Anthropometry continues to play a major role in characterising growth and nutritional status. METHODS: We developed a range of models to aid in identifying neonates at risk of malnutrition. We first adopted a logistic regression approach to screen for a composite neonatal morbidity, low and high body fat (BF%) infants. We then developed linear regression models for the estimation of neonatal fat mass as an assessment of body composition and nutritional status. RESULTS: We fitted logistic regression models combining up to four anthropometric variables to predict composite morbidity and low and high BF% neonates. The greatest area under receiver-operator characteristic curves (AUC with 95% confidence intervals (CI)) for identifying composite morbidity was 0.740 (0.63, 0.85), resulting from the combination of birthweight, length, chest and mid-thigh circumferences. The AUCs (95% CI) for identifying low and high BF% were 0.827 (0.78, 0.88) and 0.834 (0.79, 0.88), respectively. For identifying composite morbidity, BF% as measured via air displacement plethysmography showed strong predictive ability (AUC 0.786 (0.70, 0.88)), while birthweight percentiles had a lower AUC (0.695 (0.57, 0.82)). Birthweight percentiles could also identify low and high BF% neonates with AUCs of 0.792 (0.74, 0.85) and 0.834 (0.79, 0.88). We applied a sex-specific approach to anthropometric estimation of neonatal fat mass, demonstrating the influence of the testing sample size on the final model performance. CONCLUSIONS: These models display potential for further development and evaluation in LMICs to detect infants in need of further nutritional management, especially where traditional methods of risk management such as birthweight for gestational age percentiles may be variable or non-existent, or unable to detect appropriately grown, low fat newborns.

Length-free near infrared measurement of newborn malnutrition.

Under-nutrition in neonates can cause immediate mortality, impaired cognitive development and early onset adult disease. Body fat percentage measured using air-displacement-plethysmography has been found to better indicate under-nutrition than conventional birth weight percentiles. However, air-displacement-plethysmography equipment is expensive and non-portable, so is not suited for use in developing communities where the burden is often the greatest. We proposed a new body fat measurement technique using a length-free model with near-infrared spectroscopy measurements on a single site of the body - the thigh. To remove the need for length measurement, we developed a model with five discrete wavelengths and a sex parameter. The model was developed using air-displacement-plethysmography measurements in 52 neonates within 48 hours of birth. We identified instrumentation required in a low-cost LED-based screening device and incorporated a receptor device that can increase the amount of light collected. This near-infrared method may be suitable as a low cost screening tool for detecting body fat levels and monitoring nutritional interventions for malnutrition in neonates and young children in resource-constrained communities.

Ribavirin-resistant mutants of human enterovirus 71 express a high replication fidelity phenotype during growth in cell culture.

It has been shown in animal models that ribavirin-resistant poliovirus with a G64S mutation in its 3D polymerase has high replication fidelity coupled with attenuated virulence. Here, we describe the effects of mutagenesis in the human enterovirus 71 (HEV71) 3D polymerase on ribavirin resistance and replication fidelity. Seven substitutions were introduced at amino acid position 3D-G64 of a HEV71 full-length infectious cDNA clone (26M). Viable clone-derived virus populations were rescued from the G64N, G64R, and G64T mutant cDNA clones. The clone-derived G64R and G64T mutant virus populations were resistant to growth inhibition in the presence of 1,600 µM ribavirin, whereas the growth of parental 26M and the G64N mutant viruses were inhibited in the presence of 800 µM ribavirin. Nucleotide sequencing of the 2C and 3D coding regions revealed that the rate of random mutagenesis after 13 passages in the presence of 400 µM ribavirin was nearly 10 times higher in the 26M genome than in the mutant G64R virus genome. Furthermore, random mutations acquired in the 2C coding regions of 26M and G64N conferred resistance to growth inhibition in the presence of 0.5 mM guanidine, whereas the G64R and G64T mutant virus populations remained susceptible to growth inhibition by 0.5 mM guanidine. Interestingly, a S264L mutation identified in the 3D coding region of 26M after ribavirin selection was also associated with both ribavirin-resistant and high replication fidelity phenotypes. These findings are consistent with the hypothesis that the 3D-G64R, 3D-G64T, and 3D-S264L mutations confer resistance upon HEV71 to the antiviral mutagen ribavirin, coupled with a high replication fidelity phenotype during growth in cell culture.

Selection and characterisation of guanidine-resistant mutants of human enterovirus 71.

The replication of human enterovirus 71 (HEV71) in cell culture is inhibited by concentrations of guanidine that do not have an observable adverse effect on host cell metabolism. Although the HEV71 non-structural protein 2C is known to play an important role in viral RNA replication, its precise biochemical activities and structure have not been fully determined. Here we describe amino acid substitutions in HEV71 protein 2C that confer resistance to guanidine. Three guanidine-resistant virus populations were independently isolated and found to contain five mutations in protein 2C, one of which, A4657T (2C-M193L), was present in two of the independently selected populations. This mutation was introduced into a HEV71 infectious cDNA clone and was sufficient to confer complete resistance to growth inhibition in the presence of 4mM guanidine. In the first guanidine-resistant population selected, the 2C-M193L mutation occurred in association with an additional mutation, A4459G (2C-I127V), located in the putative cis-acting replication element (cre) of coding region 2C. This mutation conferred only partial guanidine resistance when introduced into the HEV71-26M infectious clone. When the 2C-I127V and 2C-M193L mutations were introduced into HEV71-26M together, the 2C-I127V mutation did not increase the level of guanidine resistance due to the 2C-M193L mutation alone. This study confirms that guanidine resistance can be readily selected in HEV71 and is attributable to mutations within protein 2C.

Mapping genetic determinants of the cell-culture growth phenotype of enterovirus 71.

Enterovirus 71 (EV71) is a member of the species Human enterovirus A within the family Picornaviridae and is a major causative agent of epidemics of hand, foot and mouth disease associated with severe neurological disease. Three EV71 genogroups, designated A, B and C, have been identified, with 75-84 % nucleotide sequence similarity between them. Two strains, EV71-26M (genogroup B) and EV71-6F (genogroup C), were found to have distinct cell-culture growth (26M, rapid; 6F, slow) and plaque-formation (26M, large; 6F, small) phenotypes. To identify the genome regions responsible for the growth phenotypes of the two strains, a series of chimeric viruses was constructed by exchanging the 5' untranslated region (UTR), P1 structural protein or P2/P3 non-structural protein gene regions plus the 3'UTR using infectious cDNA clones of both virus strains. Analysis of reciprocal virus chimeras revealed that the 5'UTRs of both strains were compatible, but not responsible for the observed phenotypes. Introduction of the EV71-6F P1 region into the EV71-26M clone resulted in a small-plaque and slow-growth phenotype similar to that of EV71-6F, whereas the reciprocal chimera displayed intermediate-growth and intermediate-sized plaque phenotypes. Introduction of the EV71-26M P2-P3-3'UTR regions into the EV71-6F clone resulted in a large-plaque and rapid-growth phenotype identical to that of strain EV71-26M, whereas the reciprocal chimera retained the background strain large-plaque phenotype. These results indicate that, although both the P1 and P2-P3-3'UTR genome regions influence the EV71 growth phenotype in cell culture, phenotype expression is dependent on specific genome-segment combinations and is not reciprocal.

The distribution of insertion sequences in the genome of Shigella flexneri strain 2457T.

Shigella flexneri, which causes shigellosis in humans, evolved from Escherichia coli. The sequencing of Shigella genomes has revealed that a large number of insertion sequence (IS) elements (over 200 elements) reside in the genome. Although the presence of these elements has been noted previously and summarized, more detailed analyses are required to understand their evolutionary significance. Here, the genome of S. flexneri strain 2457T is used to investigate the spatial distribution of IS copies around the chromosome and the location of elements with respect to genes. It is found that most IS isoforms occur essentially randomly around the genome. Two exceptions are IS91 and IS911, which appear to cluster due to local hopping. The location of IS elements with respect to genes is biased, however, revealing the action of natural selection. The non-coding regions of the genome (no more than 21%) carry disproportionally more IS elements (at least 28%) than the coding regions, implying that selection acts against insertion into genes. Of the genes disrupted by ISs, those involved in signal transduction, intracellular trafficking, and cell motility are most commonly targeted, suggesting selection against genes in these categories.