Confession Session: Learning from Others Mistakes

Accepted versio

Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

BACKGROUND: Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics. METHODOLOGY/PRINCIPAL FINDINGS: The objective of this work was to develop an alternative to conventional phage lysis tests--a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages phiA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. phiA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10(3) CFU/ml (equivalent to as few as one Y. pestis cell per 1-microl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, phiA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR. CONCLUSIONS/SIGNIFICANCE: Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria

Effect of formulation factors on in vitro transcorneal permeation of gatifloxacin from aqueous drops

The purpose of this research was to optimize the formulation factors for maximum in vitro permeation of gatifloxacin from aqueous drops through excised goat cornea and to evaluate the permeation characteristics of drug from selected marketed eyedrop formulations. Permeation studies were conducted by putting 1 mL of formulation on the cornea (0.67 cm2) fixed between the donor and receptor compartments of an all-glass modified Franz diffusion cell and measuring gatifloxacin concentration in the receptor (containing normal saline under stirring) by spectrophotometry at 291.5 nm, after 120 minutes. Raising the drug concentration of the drops increased the drug permeation but decreased the percent permeation and the in vitro ocular availability. Raising the pH of the formulation from pH 5 to 7.2 increased both the drug permeation and the in vitro ocular availability. Eyedrops containing benzalkonium chloride (BAK; 0.01% wt/vol) and disodium edetate (EDTA; 0.01% wt/vol) showed maximum permeation, followed by Zymar, BAK (0.01% wt/vol), Gatilox, Gatiquin, and Gate (statistically significantP<.05 compared with control). In vitro titration of the formulations with 0.1N NaOH indicated the presence of a buffer in Zymar (pH 6) and Gate (pH 5.8), which may cause irritation and induce lacrimation, resulting in reduced ocular availability in vivo. Thus, formulation with BAK and EDTA, which is unbuffered, has a better likelihood of being absorbed in vivo. The BAK-EDTA formulation significantly (P<.05) increased the permeation of gatifloxacin through paired excised corneas of goat, sheep, and buffalo, compared with the control formulation. The goat cornea showed the greatest increase in permeation, followed by the sheep and buffalo corneas