Gram stains: a resource for retrospective analysis of bacterial pathogens in clinical studies.

We demonstrate the feasibility of using qPCR on DNA extracted from vaginal Gram stain slides to estimate the presence and relative abundance of specific bacterial pathogens. We first tested Gram stained slides spiked with a mix of 10(8) cfu/ml of Escherichia coli and 10(5) cfu/ml of Lactobacillus acidophilus. Primers were designed for amplification of total and species-specific bacterial DNA based on 16S ribosomal gene regions. Sample DNA was pre-amplified with nearly full length 16S rDNA ribosomal gene fragment, followed by quantitative PCR with genera and species-specific 16S rDNA primers. Pre-amplification PCR increased the bacterial amounts; relative proportions of Escherichia coli and Lactobacillus recovered from spiked slides remained unchanged. We applied this method to forty two archived Gram stained slides available from a clinical trial of cerclage in pregnant women at high risk of preterm birth. We found a high correlation between Nugent scores based on bacterial morphology of Lactobacillus, Gardenerella and Mobiluncus and amounts of quantitative PCR estimated genus specific DNA (rrn copies) from Gram stained slides. Testing of a convenience sample of eight paired vaginal swabs and Gram stains freshly collected from healthy women found similar qPCR generated estimates of Lactobacillus proportions from Gram stained slides and vaginal swabs. Archived Gram stained slides collected from large scale epidemiologic and clinical studies represent a valuable, untapped resource for research on the composition of bacterial communities that colonize human mucosal surfaces.

Multiple congenital anomalies and developmental delay in a boy associated with a de novo 16p13.3 deletion.

We describe a patient with multiple congenital anomalies including tracheobronchomalacia, CT-proven metopic craniosynostosis, glandular hypospadias and severe ventral chordee, torticollis, esotropia, strabismus, fifth finger clinodactyly, hallux valgus, and global developmental delay. Using high resolution chromosomal microarray analysis, we identified a de novo deletion of 555 kb on chromosome 16p13.3, 444 kb telomeric to the CREBBP gene and 623 kb centromeric of PKD1. Review of the literature revealed numerous reports of individuals with deletions involving adjacent regions including CREBBP, but only one overlapping with this isolated region of 16p13.3. Haploinsufficiency for one or more of the 25 candidate genes in the deleted genomic region may be responsible for these clinical features. No copy number variants (CNVs) span the entire region, but several small CNVs within the 555 kb genomic region reduce the likelihood for effects due to haploinsufficiency to 18 genes.