Validation of a New PCR-Based Screening Method for Prevention of Serratia marcescens Outbreaks in the Neonatal Intensive Care Unit.

BACKGROUND: Serratia marcescens may cause severe nosocomial infections, mostly in very low birth weight infants. Since S. marcescens exhibits by far the highest adjusted incidence rate for horizontal transmission, it can cause complex outbreak situations in neonatal intensive care units. OBJECTIVE: The aim of this study was to establish a fast and highly sensitive colonization screening for prompt cohorting and barrier nursing strategies. METHODS: A probe-based duplex PCR assay targeting the 16S rRNA gene of S. marcescens was developed and validated by using 36 reference strains, 14 S. marcescens outbreak- and nonoutbreak isolates, defined by epidemiological linkage and molecular typing, and applied in 1,347 clinical specimens from 505 patients. RESULTS AND CONCLUSIONS: The novel PCR assay proved to be highly specific and had an in vitro sensitivity of 100 gene copies per reaction (∼15 bacteria). It showed a similar (in laryngeal/tracheal specimens) or even higher (in rectal/stoma swabs) in vivo sensitivity in comparison to routine microbial culture and was much quicker (<24 h vs. 2 days). By combining different oligonucleotide primers, there was robust detection of genetic variants of S. marcescens strains. PCR inhibition was low (1.6%) and observed with rectal swabs only. Cohort analysis illustrated applicability of the PCR assay as a quick tool to prevent outbreak scenarios by allowing rapid decisions on cohorting and barrier nursing. In summary, this novel molecular screening for colonization by S. marcescens is specific, highly sensitive, and substantially accelerates detection.

PCR diagnosis and characterization of Leishmania in local and imported clinical samples.

Leishmaniasis diagnosis in regions where multiple species exist should identify each species directly in the clinical sample without parasite culturing. The sensitivity of two PCR approaches which amplify part of the ssu rRNA gene and the ribosomal internal transcribed spacer (ITS), respectively, was determined using human and dog blood seeded with Leishmania promastigotes. ssu-rDNA-PCR was more sensitive than ITS1-PCR, however species identification was not possible by the former approach. When a nested ITS1-PCR was used its sensitivity equaled the ssu-rDNA-PCR. Digestion of ITS1 amplicon with the restriction enzyme HaeIII distinguished all medically relevant Leishmania species. ITS1-PCR was used to diagnose 162 local and imported suspected cases of leishmaniasis in Israel, the Palestinian Authority and Germany. 113 cases (69.7%) were positive by PCR and species identification was possible in 110 samples. Leishmania DNA was also amplified and identified at the species level from archived non-stained and Giemsa stained microscope slides.