Validation of caregiver interviews to diagnose common causes of severe neonatal illness

The objective of this study was to validate retrospective caregiver interviews for diagnosing major causes of severe neonatal illness and death. A convenience sample of 149 infants aged < 28 days with one or more suspected diagnoses of interest (low birthweight/severe malnutrition, preterm birth, birth asphyxia, birth trauma, neonatal tetanus, pneumonia, meningitis, septicaemia, diarrhoea, congenital malformation or injury) was taken from patients admitted to two hospitals in Dhaka, Bangladesh. Study paediatricians performed a standardised history and physical examination and ordered laboratory and radiographic tests according to study criteria. With a median interval of 64.5 days after death or hospital discharge, caregivers of 118 (79%) infants were interviewed about their child's illness. Using reference diagnoses based on predefined clinical and laboratory criteria, the sensitivity and specificity of particular combinations of signs (algorithms) reported by the caregivers were ascertained. Sufficient numbers of children with five reference standard diagnoses were studied to validate caregiver reports. Algorithms with sensitivity and specificity > 80% were identified for neonatal tetanus, low birthweight/severe malnutrition and preterm delivery. Algorithms with specificities > 80% for birth asphyxia and pneumonia had sensitivities < 70%, or alternatively had high sensitivity with lower specificity. In settings with limited access to medical care, retrospective caregiver interviews provide a valid means of diagnosing several of the most common causes of severe neonatal illness and death.     

Synthesis and characterization of a stable analog of the phosphorylated form of the chemotaxis protein CheY

The bacterial chemotaxis protein CheY is activated in vivo by the covalent phosphorylation of a single aspartate residue at position 57. However, this phosphate linkage is unstable (t1/2 approximately 20 s at room temperature), thereby precluding many biochemical analyses. Here we present a synthetic scheme to prepare an analog of CheY-phosphate (Che Y-P) with chemical stability of the phosphate linkage enhanced by several orders of magnitude relative to the native protein. Starting with CheY D57C, a site-specific mutant of CheY with a unique cysteine residue in place of the aspartate at position 57, two sequential disulfide exchange reactions were performed to form the final product 'CheY D57C-SPO3' with a thiophosphate moiety covalently bonded to the protein in a disulfide linkage. Mass spectral analysis showed that the desired analog was present at 70-80% of the total protein. The disulfide linkage had a t1/2 of 8 days at 4 degrees C. Biochemical characterization of CheY D57C-SPO3 included assessment of conformational properties using tryptophan fluorescence, evaluation of metal binding properties and measurement of binding interactions with the chemotaxis proteins CheZ and FliM. Despite possessing a phosphoryl group at a nearly identical location as native CheY-phosphate, the analog was unable to emulate CheY-phosphate function, thereby supporting the idea that there are very precise geometric requirements for successful CheY activation.