Ratio of neutrophilic CD64 and monocytic HLA-DR: a novel parameter in diagnosis and prognostication of neonatal sepsis

Objective: Approaches to monitoring of sepsis have traditionally relied upon the pro-inflammatory component of the sepsis response. This study evaluated the diagnostic and prognostic potential of the ratio of neutrophilic CD64 (nCD64) and monocytic HLA-DR (mHLA-DR) median fluorescence index in monitoring of neonatal sepsis. Methods: Blood from 100 neonates suspected of sepsis and 29 healthy controls was collected on clinical suspicion of sepsis, and the expression of nCD64, mHLA-DR was evaluated by Flow Cytometry; thereby, a derived parameter “Sepsis index,” SI = nCD64/mHLA-DR × 100 was estimated. Results: At day 1, sensitivity and specificity to detect sepsis using nCD64 was 73.01% and 89.18%, respectively, while for SI it was 73.01% and 72.22%, respectively. On Kaplan-Meier analysis, neonates with SI > cut-off showed a higher 30 day-mortality than those with low SI (P = 0.096). On multivariate analysis, the factor associated with mortality in our cohort was Apgar score ≤3, while SI showed a trend toward significance. Conclusions: At day1, nCD64 is useful for the diagnosis of neonatal sepsis whereas mHLA-DR is beneficial for monitoring patients at a later time point. The SI is a marker of moderate diagnostic sensitivity and supplements the current arsenal of laboratory investigations to detect neonatal sepsis. As a marker of prognosis, a high SI shows a trend towards greater mortality

Microbial Communities Associated with Marine Arthropods

The microbial ecology associated with the unaffected integuments and shell disease lesions of four species of large New England arthropods ( Cancer borealis (Jonah crab), Carcinus maenas (green crab), Limulus polyphemus (Atlantic horseshoe crab), Libinia emarginata (spider crab)) was studied. The diversity of microbial communities was identified by PCR-DGGE of bacterial 16S rRNA gene fragments. In all four species of Arthropods the composition of microbial communities in both lesions and unaffected integument surfaces were very diverse with up to 30 distinct bands evident on DGGE gels. There was not a single bacterium that was detected in all lesions and all unaffected integument samples of all four Arthropods. The results of multivariate analyses depicted that the microbial communities on unaffected integuments are dissimilar to those in lesions. Real-time PCR assays were developed for quantification of the unculturable bacteria, 'Thalassobius' sp. and Candidatus 'Homarophilus dermatus', associated with the lobster integument. A. macrocephali subsp. homaria and 'Thalassobius' sp. were found to be associated with lesion. In lesions the levels of A. macrocephali subsp. homaria and 'Thalassobius' sp. were five and three orders of magnitude higher equated to those on the unaffected integument. The abundance of 'Thalassobius' sp. in lesions was not affected by the location from where the lobsters were collected. In case of Candidatus 'Homarophilus dermatus' there were statistically more cells present on unaffected integument versus lesion, but the difference was less than one order of magnitude. The consistency in distribution of this bacterium in comparable numbers on lobsters from various locations indicated that it is a part of the normal flora of the American lobster

Real-time PCR assay for Aquimarina macrocephali subsp. homaria and its distribution in shell disease lesions of Homarus americanus, Milne-Edwards, 1837, and environmental samples

Epizootic shell disease (ESD) is causing major losses to the lobster fishery in southern New England. Potential pathogens have been identified in lesion communities, but there are currently no efficient means of detecting and quantifying their presence. A qPCR assay was developed for a key potential pathogen, Aquimarina macrocephali subsp. homaria found to be ubiquitous in ESD lesions but not the unaffected integument. Application of the assay to various samples demonstrated that A. macrocephali subsp. homaria is ubiquitous and abundant in lobster lesions, commonly associated with healthy surfaces of crabs and is scarce in water and sediment samples from southern New England suggesting the affinity of this microorganism to the Arthropod integument. The qPCR assay developed here can be applied in future in vivo and in vitro studies to better understand the ecology and role of A. macrocephali subsp.homaria. in shell disease